Exogenous hydrogen sulfide ameliorates memory dysfunction in post-stroke depressed mice by reducing NLRP3 inflammasome activation in astrocytes

Exogenous hydrogen sulfide ameliorates memory dysfunction in post-stroke depressed mice by reducing NLRP3 inflammasome activation in astrocytes | 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 Exogenous hydrogen sulfide ameliorates memory dysfunction in post-stroke depressed mice by reducing NLRP3 inflammasome activation in astrocytes Yan-nan Sun, Shu-peng Wang, Zhao-hua Guo, Yu-dong Shan, Li-li Cui, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4557185/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 Cognitive decline is common in post-stroke depression (PSD) and has been reported to be associated with oxidative stress. Hydrogen sulfide (H 2 S)—an antioxidant gas molecule—participates in producing the antioxidant glutathione, upregulating antioxidant signals, and maintaining mitochondrial integrity. In this study, we investigated the neuroprotective effects of sodium hydrosulfide (NaHS, an H 2 S donor) on cognitive impairment after PSD. After subjecting middle cerebral artery occlusion (MCAO) mice to chronic restraint stress (CRS) for 21 days, NaHS 0.1 mmol/kg was injected intraperitoneally daily for seven consecutive days. The forced swimming test (FST) and fear conditioning test (FC) were used to evaluate depression-like behavior and cognitive function. NaHS administration significantly reversed MCAO plus CRS-induced PSD and cognitive impairment, including increased immobility time, reduced context-related freezing time, elevated astrocytic pyroptosis indicated by interleukin-18 and cleaved caspase-1 in the hippocampal CA1 and CA3, downregulated oscillations in theta, and upregulated oscillations in gamma under FC conditions. H 2 S provides a new perspective for treating cognitive impairment in PSD patients. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Stroke ranked third in terms of disability and is the second most common cause of death worldwide (Das and G, 2018). Depression is one of the most prevalent side effects of strokes (Villa et al., 2018 ). One-third of the patients exhibit depression after stroke, which seriously affects treatment and prognosis. Empirical evidence has implicated organic factors in post-stroke depression (PSD), with variations in ascending monoamine systems (Li et al., 2014 ), modifications in neuroplasticity and glutamate neurotransmission (Noonan et al., 2013 ), and excessive proinflammatory cytokines (Spalletta et al., 2006 ). Nevertheless, a pathophysiological theory for PSD that incorporates these modifications into a logical explanatory framework has not yet been developed. Astrocytes are the most prevalent cell type in the nervous system and are crucial for neurotransmitter metabolism, ion balance maintenance, and nutrient delivery to neurons (Linnerbauer et al., 2020 ). The interaction between astrocytes and neurons significantly influences environmental homeostasis, plasticity, and neural information transmission in the central nervous system (Bélanger et al., 2011 ; Soto et al., 2023 ). GFAP is an important skeleton protein synthesized by astrocytes and is a specific marker of astrocytes (Middeldorp and Hol, 2011 ). Research has demonstrated that astrocyte pyroptosis is closely related to neuronal damage after PSD (Li et al., 2021 ), but the underlying mechanism remains unclear. Pyroptosis is a proinflammatory type of planned cell death (Lin and Zhang, 2017 ). Unlike apoptosis, pyroptosis occurs more rapidly and is accompanied by cell membrane rupture and the excessive release of inflammatory substances. Therefore, pyroptosis is also called "inflammatory necrosis" (Tang et al., 2023 ). Under oxidative stress, pyroptosis-induced cell death is rapid and causes significant damage to brain cells (Maiese, 2023 ). The release of inflammatory factors via pyroptosis can aggravate the inflammatory response and lead to further damage. It has been confirmed that pyroptosis plays an important role in infectious, metabolic, and nervous system diseases (Patel et al., 2017 ). Hydrogen sulfide (H 2 S) is known to be the third endogenous gas signaling molecule after carbon monoxide (CO) and nitric oxide (NO) (Song et al., 2023 ). It plays critical physiological roles in the nervous, cardiovascular, and endocrine systems. H 2 S, an antioxidant gas molecule, contributes to the production of the antioxidant glutathione, upregulates antioxidant signals, and maintains mitochondrial integrity (Kimura, 2013 ). Recently, it was suggested that H 2 S exhibits neuroprotective effects against ischemia-reperfusion injury and reduces infarct size by inducing hypothermia (Zhu et al., 2017 ). Notably, H 2 S can attenuate oxidative stress-induced injury in hippocampal neurons and inhibit cell pyroptosis (Chen et al., 2022 ). However, whether H 2 S administration alleviates cognitive dysfunction after PSD remains unclear. To determine how to alleviate pyroptosis induced by cerebral ischemic depression effectively, we aimed to study the effect of H 2 S on PSD in terms of learning and memory function changes, oxidative stress response, inflammatory response, and pyroptosis. Electroencephalography (EEG) can directly record the signals of bioelectrical activity in the brain. Researchers can acquire and analyze EEG signals to explore functional connectivity and information transmission between brain regions (Jensen and Colgin, 2007 ). Signals associated with fear, memory, and extinction are processed in brain pathways to form aversive stimulus associations. Although numerous studies have examined the individual contributions of some brain regions (Ji and Maren, 2007 ; Quirk and Mueller, 2008 ), little is known about their function as integrated systems. REM-associated theta coupling in the basolateral amygdala, hippocampus, and medial prefrontal cortex is related to successful consolidation in fear conditioning (Popa et al., 2010 ). This study explored the temporal and functional findings that theta-gamma coupling in fear extinction may be provided by combining cortical EEG and unit recordings from freely acting mice in a fear conditioning paradigm. We developed a mouse model to mimic PSD using a combination of chronic restraint stress (CRS) and middle cerebral artery occlusion (MCAO) (Zhang et al., 2021 ). This study aimed to investigate the neuroprotective mechanism of H 2 S and to assess whether it improves cognitive impairment by preventing hippocampal astrocyte pyroptosis under PSD conditions. This study provides an experimental basis for exploring new treatment strategies for PSD. Methods and materials 2.1. Ethics statement The Experimental Animal Ethics Committee of Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine in Hebei Province approved the protocols for animal husbandry and use, which were followed in all experiments with mice (CZX2022-KY-011) and performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Every effort was made to reduce the distress. Procedures that might inflict pain or discomfort were performed in a separate room when no other animals were present. Mice were administered intraperitoneal (i.p.) doses of amobarbital (ethyl carbamate, 1.8 g/kg) for anesthesia. Finally, the mice were euthanized by excessive sevoflurane inhalation. 2.2. Animals and experimental design Male C57BL/6J mice were provided by the Hebei Provincial Key Laboratory of Integrated Traditional and Western Medicine in Neurological Rehabilitation (Cangzhou, Hebei). The mice were housed in plastic cages with 12 h light and dark cycles at 22–24 ℃ with 60% humidity and ad libitum access to standard mouse chow and sterile tap water. 2.3. Groups Mice were randomly divided into two groups (n = 12 mice/group) during the first stage. Group (I) underwent a sham incision on the neck skin without obstructing the middle cerebral artery; Group (II) underwent a one-hour MCAO, and on the seventh day following MCAO, CRS was applied for 21 days (MCAO + CRS) (Fig. 1 ). Four groups of six mice each were randomly assigned to mice in the second stage. Group (I): standard saline injection as a model control (Vehicle + Sham); Group (II): plus NaHS administration (Sham + NaHS). NaHS (0.1 mmol/kg, Innochem Technology Co., Ltd. Beijing, China) was administered i.p. once daily for seven days. Group (III): MCAO and CRS treatments plus normal saline administration (MCAO + CRS + Vehicle); Group (IV): MCAO and CRS treatments plus NaHS administration (MCAO + CRS + NaHS). NaHS was dissolved in 0.5 mL of normal saline (Vehicle). In the third stage, mice after MCAO + CRS + NaHS were randomly divided into two groups (n = 3 mice/group). Group (I) received a lateral ventricular injection of nigericin (MCAO + CRS + Nigericin + NaHS); Group (II) received a lateral ventricular injection of (MCAO + CRS + Vehicle + NaHS). Nigericin was dissolved in the vehicle at 50 ng/µL. The MCAO + CRS + Nigericin + NaHS group mice were fixed in the prone position and sterilized with 10% H 2 O 2 . A hole was drilled according to the coordinates of 0.3 mm behind the anterior fonfonel and 1 mm to the right of the sagittal suture. Nigericin 250 ng (5 µL) was injected at approximately 1.5 µL/min for no less than 5 min. In the MCAO + CRS + Vehicle + NaHS group, 5 µL of an equal volume of saline was injected as described above. 2.4. Modeling The in vivo PSD mannequin was performed in two steps, covering MCAO (step 1) and melancholy (step 2). In step 1, spontaneously breathing C57BL/6 mice were maintained in the supine position, and anesthesia was induced once using 6–8% sevoflurane induction and 2–3% maintenance. A midline neck incision exposed the left common carotid and external carotid arteries under a microscope (MZ101; Mshot, Guangzhou, China). A 2 − 0 nylon monofilament (RWD Technology, Shenzhen, Guangdong Province, China) was inserted into the left interior carotid artery through the external carotid stump to hinder the foundation of the central cerebral artery. After 60 min of occlusion, the nylon monofilament was withdrawn to permit reperfusion, and the incision was sutured. Assessment using the modified Neurological Severity Score (mNSS) was performed 24 h after surgery. Mice with an mNss rating of 0–4 on day 1 and an mNss score of 10–18 on day 7 after surgery were excluded from the study. The PSD mannequin was mounted on mice with an mNss rating of 0–9 on day 7 after surgery. In Step 2, the melancholy mannequin was set up using CRS on day 7 after surgery. For CRS, mice were placed in a 50 mL EP tube for 6 h/day for 21 days. The restraint tube was made by making dispersed holes in the physique of a 50 mL centrifuge tube. A hole was created in the center of the lid to allow the tail of the mouse to be ignored. 2.5. Behavioral tests Behavioral experiments were performed for every group of mice (n = 6). On the 28th day after surgery, each mouse was individually placed in a vertical cylinder (diameter:10 cm; height: 50 cm) filled with warm water (depth: 15 cm) for 10 min for adaptation. On day 29, after surgery, mice were individually forced to swim for 6 min. The motion at some stage in the subsequent 5 min was recorded to analyze the immobility time. The formation of associative memories is crucial for the survival of an animal because it ensures adaptive behavioral responses in a constantly changing environment. The mice were allowed to rest for 1 h after forced swimming and then placed in an electric shock box (environment A) for 5 min (adaptation period), followed by 30 s of sound stimulation (85 dB, 5000 Hz). Subsequently, unavoidable plantar shock (0.6 mA) was observed for 2 s. Three sound-shock pairs were prepared and separated by 3 min. The computer software recorded the freezing time at each stage (including the adaptation period, three rounds of sound stimulation, and three rounds of intermittent period), and the percentage of freezing time (%) = freezing time (s)/total time (s × 100%). Situational fear expression detection: After 24 h of conditioned worry training, the mice were placed in a system that had received electric shocks (environment A: plantar electric fence + white light) without any stimulation, and the environment was reproduced. Freezing time was recorded within 5 min, and the percentage of freezing time was calculated. Sound cue fear expression detection: 2 h after scene fear detection, mice were transferred to a new environment (environment B: Sound stimulation was performed for 30 s after the 2 min adaptation period. The animals were removed 30 s after the end of sound stimulation. The freezing time at each stage (including adaptation, sound stimulation, and intermittent periods) was recorded, and the percentage of freezing time was calculated. The fear conditioning detection system included a fear conditioning test box and Video Freeze SOF-843 freeze time acquisition software (Med Associates Inc., USA). 2.6. Electrophysiology Three mice from each group were used for the electrophysiological experiments. The mice underwent electrophysiological testing on the 28th day after surgery, as described in a previous study (Shibata et al., 2021 ). Six 0.6 mm insulated patent leather wires, measuring 2.0 cm in length, were soldered to the pin of the socket. The patent leather was peeled off at the end of 0.5 cm to wrap the wire around the cranial nail and conduct electricity. The soldering area was insulated with hot glue. Under sevoflurane anesthesia, the head and neck were shaved after the righting reflex disappeared. The heads of the mice were sterilized and fixed with a stereotaxic apparatus (with a thermal pad on the operating table to avoid hypothermia). The skin tissue (a circle with a diameter not less than the length of the anterior and posterior fontanels) was cut off from the heads of the mice. The exposed tissue under the cranium was treated with a 3% H 2 O 2 solution to expose the bone window. Using a brain stereotaxic instrument, the coordinates of the left occipital lobe (AP-2.5, ML-2.0), right occipital lobe (AP-2.5, ML + 2.0), left prefrontal cortex (AP + 1.0, ML-1.0), and right prefrontal cortex (AP-1.0, ML-1.0) were delineated on the skull surface. A hole (approximately 1.0 mm in diameter) was drilled, and the cranial nail was screwed vertically to the surface of the brain tissue. Subsequently, an EEG electrode connection device was installed. Two electrode-connecting wires were connected to the two cranial nails in the prefrontal cortex, and the other two wires were wound around the two cranial nails in the occipital lobe as ground wires. Finally, the remaining two electrode wires were inserted into the back muscles of the mice for electromyography, and the sockets of the intracranial electrode-connecting devices were partially exposed outside the incisions on the back of the neck. After disinfection with iodophor, the cranial nails and electrode leads were adequately encapsulated using denture cement. Electrophysiology was used in the FC experiment to observe neural electrophysiological activity in mice when sound-induced fear. At the beginning of extinction training (day 2), under slight Forene anesthesia (isoflurane, 1-chloro-2,2,2 trifluoroethyldifluoromethylether), animals were connected to a swivel commutator for the recording device and after a recuperation period of 30 min, the scan started. For the EEG recordings, bandpass filtering was performed between 0.7 and 154 Hz and between 100 Hz and 13 kHz. The filtered signals were then processed in the single-channel mode using a multi-acquisition processor (NeuroStudo) system for real-time threshold setting and waveform recognition. Noise was defined as the historical activity level over 10 s. For further analysis, time stamps of neural spikes and area-doable recordings were exported to NeuroExplorer (NEX Technologies). 2.7. Immunofluorescence Immunofluorescence staining was performed on day 36 after the behavioral test (n = 6). After 6–8% anesthesia induction, the mouse aorta was perfused with saline until the crystalloid flowed from the right atrial appendage. The brain tissue was isolated after perfusion with 4% paraformaldehyde via the aorta. Following a 48-h immersion in 4% paraformaldehyde, 5 µm paraffin-embedded sections containing the hippocampus were prepared. Sections were deparaffinized with xylene and ethanol, hydrated, and boiled for 20 min at 100°C with a modified sodium citrate antigenicity restoration solution. After treatment with 1% Triton X-100 for 20 min, cells were incubated with QuickBlock™ Blocking Buffer for Immunol Staining (P0260, Shanghai Beyotime, China) for 15 min at 25°C. Sections or cells were incubated overnight at 4°C with primary mouse anti-GFAP monoclonal antibody (dilution: 1:150, GB12090-10, Servicebio, Wuhan, Hubei, China), rabbit anti-NLRP3 monoclonal antibody (dilution: 1:150, NO.K008087P, Solarbio, Beijing, China), rabbit anti-IL-18 polyclonal antibody (dilution: 1:100, NO.K101295P, Solarbio, Beijing, China), and rabbit anti-cleaved caspase-1 polyclonal antibody (dilution: 1:150, AF4022, Affinity, USA), respectively. The following day, sections were incubated with the appropriate secondary antibody mixture for 1 h and blocked by DAPI staining. All immunostaining procedures were performed under the same conditions to minimize inter-sample variability. Immunofluorescence images and motorized stages were captured using a fluorescence microscope (Conforcol microscope, SOPTOP CLSM600, Ningbo, Jiangsu, China). Immunoreactive cells were counted in 1/10 collection 40 µm coronal sections with an optical fractionator using the Ster-eolnv Investigator software program model 9 (MicroBrightField, Williston, VA). We analyzed the cell number and immunofluorescence intensity in defined areas using the ImageJ software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA), as described in our previous study (Zhang et al., 2023 ). Furthermore, Sholl analysis assessed the branch tips of specific astrocytes represented by GFAP-positive cells. The percentage of cleaved caspase-1/GFAP/DAPI triple-positive cells in the hippocampal CA1 and CA3 regions was calculated. Positive expression of cleaved caspase-1/GFAP/DAPI represents astrocyte pyroptosis. 2.8. ELISA test GFAP and interleukin-18 (IL-18) levels in the blood of mice were measured according to the manufacturer's instructions (ml002294, Mlbio, Shanghai, China; and ml001994, Mlbio, Shanghai, China). Blank, standard, and sample wells were used. The dilution solution and sample were added to the standard wells and incubated at room temperature. The optical density (OD) of each well was measured at 450 nm using a microplate reader. 2.9. Statistical analysis Sampling sizes are indicated in the figure legends (n = number of mice or cultures). Statistical analyses were performed, and graphs were plotted using GraphPad Prism (version 9). Normality was determined using the Shapiro-Wilk normality test. Results are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using a one-way analysis of variance with put up hoc Tukey's test or the Kruskal-Wallis test to determine if assumptions were no longer met. Statistical significance was set at P < 0.05. Results 2.1. NaHS can improve cognitive impairment in PSD mice In a previous study, the incidence of cognitive impairment in MCAO mice caused by CRS was approximately 87% (Kim and Diamond, 2002 ). Previous studies have demonstrated that MCAO mice exhibit depression-like behavior and cognitive impairment under CRS conditions (Li et al., 2020 ). Behavioral tests such as the forced swimming test (FST) and fear conditioning (FC) were used to explore the effects of NaHS on cognitive function in PSD mice. The FST findings revealed that the floating time of the MCAO + CRS + NaHS group was less than that of the MCAO + CRS + Vehicle group, indicating that depression in the MCAO + CRS + NaHS group was significantly improved compared to the MCAO + CRS + Vehicle group. According to the FC results, the MCAO + CRS + NaHS group exhibited superior memory compared with the MCAO + CRS + Vehicle group. Behavioral analysis revealed no significant differences between the Sham + Vehicle and Sham + NaHS groups. Furthermore, preliminary experiments (Appendix) demonstrated that both high (0.2 mmol/kg) and low (0.05 mmol/kg) doses of NaHS reduced depression-like behavior in the FST. However, the middle (0.1 mmol/kg) doses of NaHS exhibited the most obvious improvement, implying a significant therapeutic effect of NaHS on depression. 2.2. NaHS reduced astrocyte pyroptosis in the hippocampus Immunofluorescence staining was used to monitor the effects of NaHS on the hippocampal CA1 and CA3 neurons in mice. The findings revealed significant differences in improving GFAP activation in the CA1 and CA3 regions of the hippocampus among the four groups. GFAP activation significantly decreased in the MCAO + CRS + NaHS group compared to the other three groups. Significant variations were observed in the pyroptosis rates of astrocytes in the hippocampal CA1 and CA3 regions across the four groups. The pyroptosis rate of astrocytes in the CA1 and CA3 regions of the hippocampus in the MCAO + CRS + NaHS group was significantly lower than that in the MCAO + CRS + Vehicle group. Compared with the Sham group, GFAP intensity and the total number of crossings were increased in the CA1 and CA3 hippocampal regions in the MCAO + CRS + Vehicle group, and astrocytes were hyperproliferated. However, compared to the MCAO + CRS + Vehicle group, the MCAO + CRS + NaHS group exhibited significantly lower GFAP intensity and total number of crossings in the CA1 and CA3 hippocampal regions, indicating that NAHS inhibited astrogliosis and ameliorated pyroptosis. Furthermore, we examined NLRP3 expression in astrocytes and the downstream factors of NLPR3-induced pyroptosis, IL-18, and cleaved caspase-1. The cleaved caspase-1 and IL-18 expression levels were significantly reduced in the MCAO + CRS + NaHS group compared to those in the MCAO + CRS + Vehicle group. There was no significant difference in cleaved caspase-1 and IL-18 expression between the Sham + Vehicle and Sham + NaHS groups. The levels of IL-18 and cleave caspase-1 in the hippocampus of the MCAO + CRS + Vehicle, Sham + Vehicle, and Sham + NaHS groups were significantly higher than those in the MCAO + CRS + NaHS group. However, nigericin significantly reversed this decrease in cleaved caspase-1 and IL-18 levels in astrocytes. Compared to the Sham + Vehicle group, the levels of IL-18 and GFAP in the Sham + NaHS group were not significantly different, whereas IL-18 and GFAP levels were significantly increased in the MCAO + CRS + Vehicle group. Compared with the MCAO + CRS + Vehicle group, the levels of IL-8 and GFAP in the MCAO + CRS + NaHS group were significantly decreased. However, they were still superior to those in the Sham + Vehicle group. ELIZA results suggested that H 2 S could inhibit the synthesis and release of inflammatory factors in PSD mice. Under FC conditions, theta oscillations and gamma oscillations were downregulated in the MCAO + CRS mice The sound stimulus induced an obvious frozen state in the MCAO + CRS + NaHS, Sham + NaHS, and Sham + Vehicle groups during the sound test on the second day of the FC experiment. The EEG changed from recurrent paroxysmal spikes and slow waves to rhythmic bursts, the amplitude significantly higher than the background activity. However, the EEG of the MCAO + CRS + Vehicle group revealed persistent multiphasic spike slow waves throughout the sound test and the absence of a clear dividing line during sound stimulation. It is well known that theta (4–8 Hz) is linked to cognitive control, and gamma (30–150 Hz) is closely associated with cognition and memory integration and retrieval (Buzsáki and Draguhn, 2004 ; Nimmrich et al., 2015 ). Compared to the Sham group, the MCAO + CRS + Vehicle group exhibited a significant reduce in theta-gamma phase-amplitude coupling in the cortex in response to sound stimulation. Conversely, no significant difference was observed between the MCAO + CRS + NaHS and Sham groups. 2.4. Intraventricular injection of nigericin reversed the neuroprotective effects of NaHS The FST results revealed that the floating time of the MCAO + CRS + Nigericin + NaHS group was higher than that of the MCAO + CRS + Vehicle + NaHS group, indicating that nigericin reversed the beneficial effects of NaHS on depression was reversed by nigericin. FC results demonstrated that the memory level of the MCAO + CRS + Nigericin + NaHS group was lower than that of the MCAO + CRS + Vehicle + NaHS group, implying that nigericin reversed the improved cognitive function of NaHS. Immunofluorescence staining revealed that the intensity of GFAP and the total number of crossing points in the hippocampal CA1 and CA3 regions of mice in the MCAO + CRS + Vehicle + NaHS group were lower than those in the MCAO + CRS + Nigericin + NaHS group. However, astrocyte proliferation and cleaved caspase-1 and IL-18 expression were not obvious. Discussion In the present study, we observed significant cognitive impairment in a mouse model of MCAO plus restraint stress that was reversed by NaHS. In addition, NaHS reversed reactive astrogliosis and increased the expression of cleaved caspase-1 and IL-18 in the hippocampal CA1 and CA3 regions of PSD mice. Moreover, lateral ventricular injection of the NLRP3 antagonist, nigericin, partially reversed the neuroprotective effect of NaHS. FST is a reliable index for assessing the degree of depression in mice. The findings of this study indicate that i.p. injection of NaHS significantly reduced the floating time of PSD mice compared to those injected with vehicle. However, it increased the floating time in the two Sham groups of mice, indicating that NaHS may help alleviate depression following stroke. The decrease in context-dependent freezing time percentage during the fear conditioning test stage is a reliable index for evaluating memory impairment in mice. The findings demonstrated that the MCAO + CRS + NaHS group exhibited longer freezing times than the MCAO + CRS + Vehicle group, suggesting that NaHS can improve the verbal cognitive function and memory ability of PSD mice. (Dempsey et al., 2017 ). Numerous studies have reported MCAO as a typical stroke model of cerebral ischemia/reperfusion damage (Liu and McCullough, 2011 ; Rhim and Lee, 2016 ). Restrictive stress is a common form of depression (Chiba et al., 2012 ). MCAO mice demonstrate significant cognitive dysfunction and depression after restraint stress, including a decline in memory, learning ability, and social ability. In this study, MCAO was induced in mice by the thread occlusion method, and restraint stress was used to construct a depression model to explore the related mechanism. The findings of this study indicate that the MCAO + CRS + Vehicle group spent less freezing time during the FC test than the Sham + Vehicle group, which may indicate a decline in memory and cognitive performance of PSD mice. In the FST, the floating time of the MCAO + CRS + Vehicle group was higher than that of the Sham + Vehicle group, suggesting that the PSD mice were depressed. Based on these results, our PSD model was successfully developed, and cognitive impairment was induced. Immunofluorescence findings revealed decreased GFAP fluorescence intensity in the hippocampal CA1 and CA3 regions. This further confirmed that hippocampal neurons were damaged, suggesting that the model of cognitive dysfunction induced by MCAO + CRS was successfully established. Studies have indicated that depressed behavior in mice with PSD is mediated by IL-18 derived from neurons and microglia (Wu et al., 2020 ). It has been documented that H 2 S can counteract cognitive impairment caused by chronic stress in rats (Habibitabar et al., 2020 ). An increasing number of studies have demonstrated that H 2 S exerts significant neuroprotective effects on the nervous system by regulating neurogenesis (Liu et al., 2020 ) and autophagy (Kang et al., 2021 ), antagonizing oxidative stress in nerve cells (Hu et al., 2016 ; Liu et al., 2017 ), and exerting anti-inflammatory effects (Wei et al., 2014 ). Therefore, H 2 S improves chronic stress-induced pyroptosis in hippocampal cells, demonstrating its important neuroprotective effect. NaHS treatment suppressed the activation of GFAP, a specific astrocyte marker, in the hippocampus of PSD mice. Compared with the Sham + Vehicle group, the expressions of cleaved caspase-1, IL-18, and GFAP in the Sham + NaHS group did not change significantly. These results indicate that H 2 S ameliorates pyroptosis in the hippocampal cells of PSD mice. This study provides a new approach to prevent and treat PSD-related diseases. Next, we investigated whether H 2 S ameliorates hippocampal pyroptosis in PSD mice and whether it is associated with the level of related factors. Studies have revealed that H 2 S can improve chronic stress-induced pyroptosis in the hippocampus of rats by upregulating GDF11 expression during chronic stress (Ma et al., 2018 ). H 2 S has been found to improve spatial learning and memory in mice with Alzheimer's disease by inhibiting the activation of the NLRP3 inflammasome, preventing the inflammatory response, reducing neuronal damage, and avoiding the adverse effects of the continuous increase in inflammation in the body during chronic stress (Chen et al., 2024 ). Moreover, previous studies by our research group have confirmed that the exogenous H 2 S donor, NaHS, can improve hippocampal damage caused by status epilepticus (Zhuang et al., 2016 ). In addition to being highly susceptible to damage and stress responses, the hippocampus is a brain region that is closely related to behavior, depression, and mood. Because of these properties of the hippocampus, previous studies have explored changes in the hippocampal CA1 and CA3. Previous studies have suggested that activated astrocytes are involved in the pathophysiology of PSD. The present study identified decreased astrocytes and inflammatory factors, including cleaved caspase-1 and IL-18, in the CAl and CA3 of PSD mice after NaHS administration. Our findings demonstrated that NaHS not only significantly alleviated depression-like behavior in PSD mice but also exhibited an excellent neuroprotective effect against ischemia/reperfusion injury. Nigericin activates NLRP3 inflammasome. Nigericin administration to NaHS-treated PSD mice revealed a significant increase in IL-18 and cleaved caspase-1 in the hippocampus of mice, indicating that nigericin reversed the protective effect of H 2 S. Therefore, NaHS alleviates cognitive dysfunction in PSD mice, and the mechanism may be related to the activation of astrocytes and the inhibition of inflammatory responses. PSD seriously affects the physical and psychological rehabilitation and prognosis of stroke patients, and its incidence is increasing annually. There are many opinions on PSD at home and abroad, and prevention and treatment strategies are mainly focused on the use of antidepressants, such as serotonin reuptake inhibitors. Therefore, exploring the mechanisms and treatment of PSD may have long-term clinical significance. Declarations Funding No funding program supported this study. Author Contribution Sun wrote the main manuscript text and prepared figures 1-6. All authors reviewed the manuscript. Data Availability Declaration All the experimental data are stored in the database of Hebei Provincial Key Laboratory of Integrated Traditional and Western Medicine in Neurological Rehabilitation, no open access web site, more raw data please refer to the supplementary material. References Bélanger M, Allaman I, Magistretti PJ (2011) Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metabol 14:724–738 Buzsáki G, Draguhn A (2004) Neuronal oscillations in cortical networks. 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Clin Exp Pharmacol Physiol 47:302–312 Liu SY, Li D, Zeng HY, Kan LY, Zou W, Zhang P, Gu HF, Tang XQ (2017) Hydrogen Sulfide Inhibits Chronic Unpredictable Mild Stress-Induced Depressive-Like Behavior by Upregulation of Sirt-1: Involvement in Suppression of Hippocampal Endoplasmic Reticulum Stress. Int J Neuropsychopharmacol 20:867–876 Ma J, Zhang L, Niu T, Ai C, Jia G, Jin X, Wen L, Zhang K, Zhang Q, Li C (2018) Growth differentiation factor 11 improves neurobehavioral recovery and stimulates angiogenesis in rats subjected to cerebral ischemia/reperfusion. Brain Res Bull 139:38–47 Maiese K (2023) The impact of aging and oxidative stress in metabolic and nervous system disorders: programmed cell death and molecular signal transduction crosstalk. Front Immunol 14:1273570 Middeldorp J, Hol EM (2011) GFAP in health and disease. Prog Neurobiol 93:421–443 Nimmrich V, Draguhn A, Axmacher N (2015) Neuronal Network Oscillations in Neurodegenerative Diseases. 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International journal of molecular sciences 24 Villa RF, Ferrari F, Moretti A (2018) Post-stroke depression: Mechanisms and pharmacological treatment. Pharmacol Ther 184:131–144 Wei HJ, Li X, Tang XQ (2014) Therapeutic benefits of H₂S in Alzheimer's disease. J Clin neuroscience: official J Neurosurgical Soc Australasia 21:1665–1669 Wu D, Zhang G, Zhao C, Yang Y, Miao Z, Xu X (2020) Interleukin-18 from neurons and microglia mediates depressive behaviors in mice with post-stroke depression. Brain, behavior, and immunity 88 , 411–420 Zhang LM, Wu ZY, Liu JZ, Li Y, Lv JM, Wang LY, Shan YD, Song RX, Miao HT, Zhang W et al (2023) Subanesthetic dose of S-ketamine improved cognitive dysfunction via the inhibition of hippocampal astrocytosis in a mouse model of post-stroke chronic stress. J Psychiatr Res 158:1–14 Zhang LM, Zhang DX, Zheng WC, Hu JS, Fu L, Li Y, Xin Y, Wang XP (2021) CORM-3 exerts a neuroprotective effect in a rodent model of traumatic brain injury via the bidirectional gut-brain interactions. Exp Neurol 341:113683 Zhu Y, Shui M, Liu X, Hu W, Wang Y (2017) Increased autophagic degradation contributes to the neuroprotection of hydrogen sulfide against cerebral ischemia/reperfusion injury. Metab Brain Dis 32:1449–1458 Zhuang F, Zhou X, Li H, Yang X, Dong Z, Zhou W, Chen J (2016) Hydrogen Sulfide Promotes Learning and Memory and Suppresses Proinflammatory Cytokines in Repetitive Febrile Seizures. Neuroimmunomodulation 23 , 271–277 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx 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-4557185","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":320650214,"identity":"8959b30f-3b09-465a-8fb2-cb9b7ff6de12","order_by":0,"name":"Yan-nan Sun","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yan-nan","middleName":"","lastName":"Sun","suffix":""},{"id":320650215,"identity":"93c92e77-efae-43de-ba03-8d49ea793eb0","order_by":1,"name":"Shu-peng Wang","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shu-peng","middleName":"","lastName":"Wang","suffix":""},{"id":320650216,"identity":"07376f65-cd02-4a7f-8268-81a80dc621e3","order_by":2,"name":"Zhao-hua Guo","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhao-hua","middleName":"","lastName":"Guo","suffix":""},{"id":320650217,"identity":"81a79980-6981-4f5e-b949-a08a60f2c43c","order_by":3,"name":"Yu-dong Shan","email":"","orcid":"","institution":"Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu-dong","middleName":"","lastName":"Shan","suffix":""},{"id":320650218,"identity":"e194f5db-5d66-443a-a8ff-4d70d863c1b3","order_by":4,"name":"Li-li Cui","email":"","orcid":"","institution":"The Second Affiliated Hospital of Hebei Medical University, Shijiazhuang, China","correspondingAuthor":false,"prefix":"","firstName":"Li-li","middleName":"","lastName":"Cui","suffix":""},{"id":320650219,"identity":"15c7e279-bc4a-4137-828e-8bedf61544b6","order_by":5,"name":"Lu-chan Yan","email":"","orcid":"","institution":"Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lu-chan","middleName":"","lastName":"Yan","suffix":""},{"id":320650220,"identity":"231638ec-9ea1-454d-9547-9d7093936200","order_by":6,"name":"Li-min Zhang","email":"","orcid":"","institution":"Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine","correspondingAuthor":false,"prefix":"","firstName":"Li-min","middleName":"","lastName":"Zhang","suffix":""},{"id":320650221,"identity":"448e46cd-593a-4a93-82a5-187616ecee16","order_by":7,"name":"Bao-dong Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYBACPgkGAwYeMJP5wIEPP4jQwgbVIgFkJh6c2UOaFh7jwxxsxGiRbt7A8KbCro5f+syHw0DN8vxiBwhokTlWwDjnTLKEZF/uhsMFFgyGM2cnEHJYjgEzb9sBCYMzvBsOz+BhSDC4TbwWngeHedhI1MJApBaoXyRn9rAZAANZgrBf+KEhxs/Pw/z4w4cfNvL80gS0AAE7cpRLEFQ+CkbBKBgFo4AIAACLDTw9bTC23AAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine","correspondingAuthor":true,"prefix":"","firstName":"Bao-dong","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-10 09:48:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4557185/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4557185/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60200174,"identity":"8c97d7d9-298a-43fd-a0ff-5532187c0647","added_by":"auto","created_at":"2024-07-13 02:31:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94983,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental schematic diagram (generated using BioRender.com).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/a3f303e582429051233f3e27.png"},{"id":60200901,"identity":"f3514982-f937-42ad-baf7-986298ba2691","added_by":"auto","created_at":"2024-07-13 02:39:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143208,"visible":true,"origin":"","legend":"\u003cp\u003eNaHS improves depressive behavior and cognitive dysfunction in mice with PSD (A) Floating time in FST. Data are presented as the mean ± SD (n = 6 mice). (B) Relative value of sound association in the FC. Data are presented as the mean ± SD (n = 6 mice).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/77d17b4e9a2d5f217ce055ee.png"},{"id":60200176,"identity":"bae90527-5ce2-4ac7-aa99-47c813e8d0c3","added_by":"auto","created_at":"2024-07-13 02:31:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":990700,"visible":true,"origin":"","legend":"\u003cp\u003eGFAP-IL-18 baseline in the CA1 and CA3 regions of the four groups of mice. (A)Representative photomicrographs. Scale bar = 20 µm. (B) Mean fluorescence density of GFAP in each group. Data are presented as the mean ± SD (n = 6 mice/group). (C) GFAP-IL-18 positive cell rates in four groups of mice. Data are presented as the mean ± SD (n = 6 mice). (D)Representative sholl [ED1] analysis photograph of GFAP. Scale bar = 5 µm. (E) The total number of GFAP intersections in the four groups of mice. Data are presented as the mean ± SD (n = 6 mice). (F) Elisa, IL-18 and GFAP concentrations in mice serum.\u003c/p\u003e\n\u003cp\u003eKindly check this, no such analysis is mentioned in the article\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/ab2e86185c2b39001634f9ce.png"},{"id":60200177,"identity":"37af26fd-f33c-4651-9b7b-f2cb56f36a29","added_by":"auto","created_at":"2024-07-13 02:31:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":478153,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophysiological results for the four groups of mice. (A) Power spectrum of the mouse EEG data in the theta band. (B) Mean theta power spectral density during sound stimulation in the FC experiments. Data are presented as the mean ± SD (n = 6 mice/group). (C) Theta–gamma coupling plots of the four groups of mice.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/e46b4b5c8e91a5eb1d7ac5b4.png"},{"id":60200179,"identity":"b99b8f9f-76aa-40d8-bb72-b2f900083011","added_by":"auto","created_at":"2024-07-13 02:31:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":95956,"visible":true,"origin":"","legend":"\u003cp\u003eIntraventricular injection of nigericin antagonized the effect of NaHS on depressive behavior and cognitive dysfunction in mice with PSD. (A) Floating time in FST. Data are presented as the mean ± SD (n = 3 mice). (B) Relative value of sound association in the FC. Data are presented as the mean ± SD (n = 3 mice).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/f4cba9e054f8afdf39e67993.png"},{"id":60200180,"identity":"e53b8cf9-ba2b-45ee-9740-7d0234e0ef28","added_by":"auto","created_at":"2024-07-13 02:31:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":311835,"visible":true,"origin":"","legend":"\u003cp\u003eGFAP-IL-18 baseline in the CA1 and CA3 regions of the two groups of mice. (A) Representative photomicrographs. Scale bar = 20 µm. (B) Mean fluorescence density of GFAP in each group. Data are presented as the mean ± SD (n = 3 mice/group). (C) GFAP-IL-18 positive cell rates in four groups of mice. Data are presented as the mean ± SD (n = 3 mice).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/f7d36050e33f0406bb7593e6.png"},{"id":60824497,"identity":"276cf784-983c-4364-a0c4-40fc8f058ac9","added_by":"auto","created_at":"2024-07-22 13:48:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2618787,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/5bd6ffc6-6280-465d-9b90-7cac00b35c8f.pdf"},{"id":60200181,"identity":"9f12747c-e6c0-44e5-b219-2b71a922bf0f","added_by":"auto","created_at":"2024-07-13 02:31:42","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":32119,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4557185/v1/b317e3b756453102645a5599.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exogenous hydrogen sulfide ameliorates memory dysfunction in post-stroke depressed mice by reducing NLRP3 inflammasome activation in astrocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStroke ranked third in terms of disability and is the second most common cause of death worldwide (Das and G, 2018). Depression is one of the most prevalent side effects of strokes (Villa et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One-third of the patients exhibit depression after stroke, which seriously affects treatment and prognosis. Empirical evidence has implicated organic factors in post-stroke depression (PSD), with variations in ascending monoamine systems (Li et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), modifications in neuroplasticity and glutamate neurotransmission (Noonan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and excessive proinflammatory cytokines (Spalletta et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Nevertheless, a pathophysiological theory for PSD that incorporates these modifications into a logical explanatory framework has not yet been developed.\u003c/p\u003e \u003cp\u003eAstrocytes are the most prevalent cell type in the nervous system and are crucial for neurotransmitter metabolism, ion balance maintenance, and nutrient delivery to neurons (Linnerbauer et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The interaction between astrocytes and neurons significantly influences environmental homeostasis, plasticity, and neural information transmission in the central nervous system (B\u0026eacute;langer et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Soto et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). GFAP is an important skeleton protein synthesized by astrocytes and is a specific marker of astrocytes (Middeldorp and Hol, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Research has demonstrated that astrocyte pyroptosis is closely related to neuronal damage after PSD (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but the underlying mechanism remains unclear. Pyroptosis is a proinflammatory type of planned cell death (Lin and Zhang, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Unlike apoptosis, pyroptosis occurs more rapidly and is accompanied by cell membrane rupture and the excessive release of inflammatory substances. Therefore, pyroptosis is also called \"inflammatory necrosis\" (Tang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Under oxidative stress, pyroptosis-induced cell death is rapid and causes significant damage to brain cells (Maiese, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The release of inflammatory factors via pyroptosis can aggravate the inflammatory response and lead to further damage. It has been confirmed that pyroptosis plays an important role in infectious, metabolic, and nervous system diseases (Patel et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) is known to be the third endogenous gas signaling molecule after carbon monoxide (CO) and nitric oxide (NO) (Song et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It plays critical physiological roles in the nervous, cardiovascular, and endocrine systems. H\u003csub\u003e2\u003c/sub\u003eS, an antioxidant gas molecule, contributes to the production of the antioxidant glutathione, upregulates antioxidant signals, and maintains mitochondrial integrity (Kimura, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Recently, it was suggested that H\u003csub\u003e2\u003c/sub\u003eS exhibits neuroprotective effects against ischemia-reperfusion injury and reduces infarct size by inducing hypothermia (Zhu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Notably, H\u003csub\u003e2\u003c/sub\u003eS can attenuate oxidative stress-induced injury in hippocampal neurons and inhibit cell pyroptosis (Chen et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, whether H\u003csub\u003e2\u003c/sub\u003eS administration alleviates cognitive dysfunction after PSD remains unclear. To determine how to alleviate pyroptosis induced by cerebral ischemic depression effectively, we aimed to study the effect of H\u003csub\u003e2\u003c/sub\u003eS on PSD in terms of learning and memory function changes, oxidative stress response, inflammatory response, and pyroptosis.\u003c/p\u003e \u003cp\u003eElectroencephalography (EEG) can directly record the signals of bioelectrical activity in the brain. Researchers can acquire and analyze EEG signals to explore functional connectivity and information transmission between brain regions (Jensen and Colgin, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Signals associated with fear, memory, and extinction are processed in brain pathways to form aversive stimulus associations. Although numerous studies have examined the individual contributions of some brain regions (Ji and Maren, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Quirk and Mueller, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), little is known about their function as integrated systems. REM-associated theta coupling in the basolateral amygdala, hippocampus, and medial prefrontal cortex is related to successful consolidation in fear conditioning (Popa et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This study explored the temporal and functional findings that theta-gamma coupling in fear extinction may be provided by combining cortical EEG and unit recordings from freely acting mice in a fear conditioning paradigm.\u003c/p\u003e \u003cp\u003eWe developed a mouse model to mimic PSD using a combination of chronic restraint stress (CRS) and middle cerebral artery occlusion (MCAO) (Zhang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study aimed to investigate the neuroprotective mechanism of H\u003csub\u003e2\u003c/sub\u003eS and to assess whether it improves cognitive impairment by preventing hippocampal astrocyte pyroptosis under PSD conditions. This study provides an experimental basis for exploring new treatment strategies for PSD.\u003c/p\u003e "},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics statement\u003c/h2\u003e \u003cp\u003e The Experimental Animal Ethics Committee of Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine in Hebei Province approved the protocols for animal husbandry and use, which were followed in all experiments with mice (CZX2022-KY-011) and performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Every effort was made to reduce the distress. Procedures that might inflict pain or discomfort were performed in a separate room when no other animals were present. Mice were administered intraperitoneal (i.p.) doses of amobarbital (ethyl carbamate, 1.8 g/kg) for anesthesia. Finally, the mice were euthanized by excessive sevoflurane inhalation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animals and experimental design\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice were provided by the Hebei Provincial Key Laboratory of Integrated Traditional and Western Medicine in Neurological Rehabilitation (Cangzhou, Hebei). The mice were housed in plastic cages with 12 h light and dark cycles at 22\u0026ndash;24 ℃ with 60% humidity and ad libitum access to standard mouse chow and sterile tap water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Groups\u003c/h2\u003e \u003cp\u003eMice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;12 mice/group) during the first stage. Group (I) underwent a sham incision on the neck skin without obstructing the middle cerebral artery; Group (II) underwent a one-hour MCAO, and on the seventh day following MCAO, CRS was applied for 21 days (MCAO\u0026thinsp;+\u0026thinsp;CRS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Four groups of six mice each were randomly assigned to mice in the second stage. Group (I): standard saline injection as a model control (Vehicle\u0026thinsp;+\u0026thinsp;Sham); Group (II): plus NaHS administration (Sham\u0026thinsp;+\u0026thinsp;NaHS). NaHS (0.1 mmol/kg, Innochem Technology Co., Ltd. Beijing, China) was administered i.p. once daily for seven days. Group (III): MCAO and CRS treatments plus normal saline administration (MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle); Group (IV): MCAO and CRS treatments plus NaHS administration (MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS). NaHS was dissolved in 0.5 mL of normal saline (Vehicle). In the third stage, mice after MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;3 mice/group). Group (I) received a lateral ventricular injection of nigericin (MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Nigericin\u0026thinsp;+\u0026thinsp;NaHS); Group (II) received a lateral ventricular injection of (MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle\u0026thinsp;+\u0026thinsp;NaHS). Nigericin was dissolved in the vehicle at 50 ng/\u0026micro;L. The MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Nigericin\u0026thinsp;+\u0026thinsp;NaHS group mice were fixed in the prone position and sterilized with 10% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. A hole was drilled according to the coordinates of 0.3 mm behind the anterior fonfonel and 1 mm to the right of the sagittal suture. Nigericin 250 ng (5 \u0026micro;L) was injected at approximately 1.5 \u0026micro;L/min for no less than 5 min. In the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle\u0026thinsp;+\u0026thinsp;NaHS group, 5 \u0026micro;L of an equal volume of saline was injected as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Modeling\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e PSD mannequin was performed in two steps, covering MCAO (step 1) and melancholy (step 2). In step 1, spontaneously breathing C57BL/6 mice were maintained in the supine position, and anesthesia was induced once using 6\u0026ndash;8% sevoflurane induction and 2\u0026ndash;3% maintenance. A midline neck incision exposed the left common carotid and external carotid arteries under a microscope (MZ101; Mshot, Guangzhou, China). A 2\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon monofilament (RWD Technology, Shenzhen, Guangdong Province, China) was inserted into the left interior carotid artery through the external carotid stump to hinder the foundation of the central cerebral artery. After 60 min of occlusion, the nylon monofilament was withdrawn to permit reperfusion, and the incision was sutured. Assessment using the modified Neurological Severity Score (mNSS) was performed 24 h after surgery. Mice with an mNss rating of 0\u0026ndash;4 on day 1 and an mNss score of 10\u0026ndash;18 on day 7 after surgery were excluded from the study. The PSD mannequin was mounted on mice with an mNss rating of 0\u0026ndash;9 on day 7 after surgery. In Step 2, the melancholy mannequin was set up using CRS on day 7 after surgery. For CRS, mice were placed in a 50 mL EP tube for 6 h/day for 21 days. The restraint tube was made by making dispersed holes in the physique of a 50 mL centrifuge tube. A hole was created in the center of the lid to allow the tail of the mouse to be ignored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Behavioral tests\u003c/h2\u003e \u003cp\u003eBehavioral experiments were performed for every group of mice (n\u0026thinsp;=\u0026thinsp;6). On the 28th day after surgery, each mouse was individually placed in a vertical cylinder (diameter:10 cm; height: 50 cm) filled with warm water (depth: 15 cm) for 10 min for adaptation. On day 29, after surgery, mice were individually forced to swim for 6 min. The motion at some stage in the subsequent 5 min was recorded to analyze the immobility time. The formation of associative memories is crucial for the survival of an animal because it ensures adaptive behavioral responses in a constantly changing environment. The mice were allowed to rest for 1 h after forced swimming and then placed in an electric shock box (environment A) for 5 min (adaptation period), followed by 30 s of sound stimulation (85 dB, 5000 Hz). Subsequently, unavoidable plantar shock (0.6 mA) was observed for 2 s. Three sound-shock pairs were prepared and separated by 3 min. The computer software recorded the freezing time at each stage (including the adaptation period, three rounds of sound stimulation, and three rounds of intermittent period), and the percentage of freezing time (%)\u0026thinsp;=\u0026thinsp;freezing time (s)/total time (s \u0026times; 100%). Situational fear expression detection: After 24 h of conditioned worry training, the mice were placed in a system that had received electric shocks (environment A: plantar electric fence\u0026thinsp;+\u0026thinsp;white light) without any stimulation, and the environment was reproduced. Freezing time was recorded within 5 min, and the percentage of freezing time was calculated.\u003c/p\u003e \u003cp\u003eSound cue fear expression detection: 2 h after scene fear detection, mice were transferred to a new environment (environment B: Sound stimulation was performed for 30 s after the 2 min adaptation period. The animals were removed 30 s after the end of sound stimulation. The freezing time at each stage (including adaptation, sound stimulation, and intermittent periods) was recorded, and the percentage of freezing time was calculated. The fear conditioning detection system included a fear conditioning test box and Video Freeze SOF-843 freeze time acquisition software (Med Associates Inc., USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Electrophysiology\u003c/h2\u003e \u003cp\u003eThree mice from each group were used for the electrophysiological experiments. The mice underwent electrophysiological testing on the 28th day after surgery, as described in a previous study (Shibata et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Six 0.6 mm insulated patent leather wires, measuring 2.0 cm in length, were soldered to the pin of the socket. The patent leather was peeled off at the end of 0.5 cm to wrap the wire around the cranial nail and conduct electricity. The soldering area was insulated with hot glue.\u003c/p\u003e \u003cp\u003eUnder sevoflurane anesthesia, the head and neck were shaved after the righting reflex disappeared. The heads of the mice were sterilized and fixed with a stereotaxic apparatus (with a thermal pad on the operating table to avoid hypothermia). The skin tissue (a circle with a diameter not less than the length of the anterior and posterior fontanels) was cut off from the heads of the mice. The exposed tissue under the cranium was treated with a 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution to expose the bone window. Using a brain stereotaxic instrument, the coordinates of the left occipital lobe (AP-2.5, ML-2.0), right occipital lobe (AP-2.5, ML\u0026thinsp;+\u0026thinsp;2.0), left prefrontal cortex (AP\u0026thinsp;+\u0026thinsp;1.0, ML-1.0), and right prefrontal cortex (AP-1.0, ML-1.0) were delineated on the skull surface. A hole (approximately 1.0 mm in diameter) was drilled, and the cranial nail was screwed vertically to the surface of the brain tissue. Subsequently, an EEG electrode connection device was installed. Two electrode-connecting wires were connected to the two cranial nails in the prefrontal cortex, and the other two wires were wound around the two cranial nails in the occipital lobe as ground wires. Finally, the remaining two electrode wires were inserted into the back muscles of the mice for electromyography, and the sockets of the intracranial electrode-connecting devices were partially exposed outside the incisions on the back of the neck. After disinfection with iodophor, the cranial nails and electrode leads were adequately encapsulated using denture cement.\u003c/p\u003e \u003cp\u003eElectrophysiology was used in the FC experiment to observe neural electrophysiological activity in mice when sound-induced fear. At the beginning of extinction training (day 2), under slight Forene anesthesia (isoflurane, 1-chloro-2,2,2 trifluoroethyldifluoromethylether), animals were connected to a swivel commutator for the recording device and after a recuperation period of 30 min, the scan started.\u003c/p\u003e \u003cp\u003eFor the EEG recordings, bandpass filtering was performed between 0.7 and 154 Hz and between 100 Hz and 13 kHz. The filtered signals were then processed in the single-channel mode using a multi-acquisition processor (NeuroStudo) system for real-time threshold setting and waveform recognition. Noise was defined as the historical activity level over 10 s. For further analysis, time stamps of neural spikes and area-doable recordings were exported to NeuroExplorer (NEX Technologies).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Immunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed on day 36 after the behavioral test (n\u0026thinsp;=\u0026thinsp;6). After 6\u0026ndash;8% anesthesia induction, the mouse aorta was perfused with saline until the crystalloid flowed from the right atrial appendage. The brain tissue was isolated after perfusion with 4% paraformaldehyde via the aorta. Following a 48-h immersion in 4% paraformaldehyde, 5 \u0026micro;m paraffin-embedded sections containing the hippocampus were prepared. Sections were deparaffinized with xylene and ethanol, hydrated, and boiled for 20 min at 100\u0026deg;C with a modified sodium citrate antigenicity restoration solution. After treatment with 1% Triton X-100 for 20 min, cells were incubated with QuickBlock\u0026trade; Blocking Buffer for Immunol Staining (P0260, Shanghai Beyotime, China) for 15 min at 25\u0026deg;C. Sections or cells were incubated overnight at 4\u0026deg;C with primary mouse anti-GFAP monoclonal antibody (dilution: 1:150, GB12090-10, Servicebio, Wuhan, Hubei, China), rabbit anti-NLRP3 monoclonal antibody (dilution: 1:150, NO.K008087P, Solarbio, Beijing, China), rabbit anti-IL-18 polyclonal antibody (dilution: 1:100, NO.K101295P, Solarbio, Beijing, China), and rabbit anti-cleaved caspase-1 polyclonal antibody (dilution: 1:150, AF4022, Affinity, USA), respectively. The following day, sections were incubated with the appropriate secondary antibody mixture for 1 h and blocked by DAPI staining. All immunostaining procedures were performed under the same conditions to minimize inter-sample variability. Immunofluorescence images and motorized stages were captured using a fluorescence microscope (Conforcol microscope, SOPTOP CLSM600, Ningbo, Jiangsu, China). Immunoreactive cells were counted in 1/10 collection 40 \u0026micro;m coronal sections with an optical fractionator using the Ster-eolnv Investigator software program model 9 (MicroBrightField, Williston, VA). We analyzed the cell number and immunofluorescence intensity in defined areas using the ImageJ software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA), as described in our previous study (Zhang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, Sholl analysis assessed the branch tips of specific astrocytes represented by GFAP-positive cells. The percentage of cleaved caspase-1/GFAP/DAPI triple-positive cells in the hippocampal CA1 and CA3 regions was calculated. Positive expression of cleaved caspase-1/GFAP/DAPI represents astrocyte pyroptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8. ELISA test\u003c/h2\u003e \u003cp\u003eGFAP and interleukin-18 (IL-18) levels in the blood of mice were measured according to the manufacturer's instructions (ml002294, Mlbio, Shanghai, China; and ml001994, Mlbio, Shanghai, China). Blank, standard, and sample wells were used. The dilution solution and sample were added to the standard wells and incubated at room temperature. The optical density (OD) of each well was measured at 450 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e \u003cp\u003eSampling sizes are indicated in the figure legends (n\u0026thinsp;=\u0026thinsp;number of mice or cultures). Statistical analyses were performed, and graphs were plotted using GraphPad Prism (version 9). Normality was determined using the Shapiro-Wilk normality test. Results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using a one-way analysis of variance with put up hoc Tukey's test or the Kruskal-Wallis test to determine if assumptions were no longer met. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.1. NaHS can improve cognitive impairment in PSD mice\u003c/h2\u003e \u003cp\u003eIn a previous study, the incidence of cognitive impairment in MCAO mice caused by CRS was approximately 87% (Kim and Diamond, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Previous studies have demonstrated that MCAO mice exhibit depression-like behavior and cognitive impairment under CRS conditions (Li et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Behavioral tests such as the forced swimming test (FST) and fear conditioning (FC) were used to explore the effects of NaHS on cognitive function in PSD mice. The FST findings revealed that the floating time of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group was less than that of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group, indicating that depression in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group was significantly improved compared to the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group. According to the FC results, the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group exhibited superior memory compared with the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group. Behavioral analysis revealed no significant differences between the Sham\u0026thinsp;+\u0026thinsp;Vehicle and Sham\u0026thinsp;+\u0026thinsp;NaHS groups. Furthermore, preliminary experiments (Appendix) demonstrated that both high (0.2 mmol/kg) and low (0.05 mmol/kg) doses of NaHS reduced depression-like behavior in the FST. However, the middle (0.1 mmol/kg) doses of NaHS exhibited the most obvious improvement, implying a significant therapeutic effect of NaHS on depression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.2. NaHS reduced astrocyte pyroptosis in the hippocampus\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was used to monitor the effects of NaHS on the hippocampal CA1 and CA3 neurons in mice. The findings revealed significant differences in improving GFAP activation in the CA1 and CA3 regions of the hippocampus among the four groups. GFAP activation significantly decreased in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group compared to the other three groups. Significant variations were observed in the pyroptosis rates of astrocytes in the hippocampal CA1 and CA3 regions across the four groups. The pyroptosis rate of astrocytes in the CA1 and CA3 regions of the hippocampus in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group was significantly lower than that in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group. Compared with the Sham group, GFAP intensity and the total number of crossings were increased in the CA1 and CA3 hippocampal regions in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group, and astrocytes were hyperproliferated. However, compared to the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group, the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group exhibited significantly lower GFAP intensity and total number of crossings in the CA1 and CA3 hippocampal regions, indicating that NAHS inhibited astrogliosis and ameliorated pyroptosis. Furthermore, we examined NLRP3 expression in astrocytes and the downstream factors of NLPR3-induced pyroptosis, IL-18, and cleaved caspase-1. The cleaved caspase-1 and IL-18 expression levels were significantly reduced in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group compared to those in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group. There was no significant difference in cleaved caspase-1 and IL-18 expression between the Sham\u0026thinsp;+\u0026thinsp;Vehicle and Sham\u0026thinsp;+\u0026thinsp;NaHS groups. The levels of IL-18 and cleave caspase-1 in the hippocampus of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle, Sham\u0026thinsp;+\u0026thinsp;Vehicle, and Sham\u0026thinsp;+\u0026thinsp;NaHS groups were significantly higher than those in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group. However, nigericin significantly reversed this decrease in cleaved caspase-1 and IL-18 levels in astrocytes.\u003c/p\u003e \u003cp\u003eCompared to the Sham\u0026thinsp;+\u0026thinsp;Vehicle group, the levels of IL-18 and GFAP in the Sham\u0026thinsp;+\u0026thinsp;NaHS group were not significantly different, whereas IL-18 and GFAP levels were significantly increased in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group. Compared with the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group, the levels of IL-8 and GFAP in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group were significantly decreased. However, they were still superior to those in the Sham\u0026thinsp;+\u0026thinsp;Vehicle group. ELIZA results suggested that H\u003csub\u003e2\u003c/sub\u003eS could inhibit the synthesis and release of inflammatory factors in PSD mice.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eUnder FC conditions, theta oscillations and gamma oscillations were downregulated in the MCAO\u0026thinsp;+\u0026thinsp;CRS mice\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThe sound stimulus induced an obvious frozen state in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS, Sham\u0026thinsp;+\u0026thinsp;NaHS, and Sham\u0026thinsp;+\u0026thinsp;Vehicle groups during the sound test on the second day of the FC experiment. The EEG changed from recurrent paroxysmal spikes and slow waves to rhythmic bursts, the amplitude significantly higher than the background activity. However, the EEG of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group revealed persistent multiphasic spike slow waves throughout the sound test and the absence of a clear dividing line during sound stimulation.\u003c/p\u003e \u003cp\u003eIt is well known that theta (4\u0026ndash;8 Hz) is linked to cognitive control, and gamma (30\u0026ndash;150 Hz) is closely associated with cognition and memory integration and retrieval (Buzs\u0026aacute;ki and Draguhn, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nimmrich et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Compared to the Sham group, the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group exhibited a significant reduce in theta-gamma phase-amplitude coupling in the cortex in response to sound stimulation. Conversely, no significant difference was observed between the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS and Sham groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Intraventricular injection of nigericin reversed the neuroprotective effects of NaHS\u003c/h2\u003e \u003cp\u003eThe FST results revealed that the floating time of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Nigericin\u0026thinsp;+\u0026thinsp;NaHS group was higher than that of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle\u0026thinsp;+\u0026thinsp;NaHS group, indicating that nigericin reversed the beneficial effects of NaHS on depression was reversed by nigericin. FC results demonstrated that the memory level of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Nigericin\u0026thinsp;+\u0026thinsp;NaHS group was lower than that of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle\u0026thinsp;+\u0026thinsp;NaHS group, implying that nigericin reversed the improved cognitive function of NaHS.\u003c/p\u003e \u003cp\u003eImmunofluorescence staining revealed that the intensity of GFAP and the total number of crossing points in the hippocampal CA1 and CA3 regions of mice in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle\u0026thinsp;+\u0026thinsp;NaHS group were lower than those in the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Nigericin\u0026thinsp;+\u0026thinsp;NaHS group. However, astrocyte proliferation and cleaved caspase-1 and IL-18 expression were not obvious.\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eIn the present study, we observed significant cognitive impairment in a mouse model of MCAO plus restraint stress that was reversed by NaHS. In addition, NaHS reversed reactive astrogliosis and increased the expression of cleaved caspase-1 and IL-18 in the hippocampal CA1 and CA3 regions of PSD mice. Moreover, lateral ventricular injection of the NLRP3 antagonist, nigericin, partially reversed the neuroprotective effect of NaHS. FST is a reliable index for assessing the degree of depression in mice. The findings of this study indicate that i.p. injection of NaHS significantly reduced the floating time of PSD mice compared to those injected with vehicle. However, it increased the floating time in the two Sham groups of mice, indicating that NaHS may help alleviate depression following stroke. The decrease in context-dependent freezing time percentage during the fear conditioning test stage is a reliable index for evaluating memory impairment in mice. The findings demonstrated that the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;NaHS group exhibited longer freezing times than the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group, suggesting that NaHS can improve the verbal cognitive function and memory ability of PSD mice. (Dempsey et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNumerous studies have reported MCAO as a typical stroke model of cerebral ischemia/reperfusion damage (Liu and McCullough, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rhim and Lee, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Restrictive stress is a common form of depression (Chiba et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). MCAO mice demonstrate significant cognitive dysfunction and depression after restraint stress, including a decline in memory, learning ability, and social ability. In this study, MCAO was induced in mice by the thread occlusion method, and restraint stress was used to construct a depression model to explore the related mechanism. The findings of this study indicate that the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group spent less freezing time during the FC test than the Sham\u0026thinsp;+\u0026thinsp;Vehicle group, which may indicate a decline in memory and cognitive performance of PSD mice. In the FST, the floating time of the MCAO\u0026thinsp;+\u0026thinsp;CRS\u0026thinsp;+\u0026thinsp;Vehicle group was higher than that of the Sham\u0026thinsp;+\u0026thinsp;Vehicle group, suggesting that the PSD mice were depressed. Based on these results, our PSD model was successfully developed, and cognitive impairment was induced. Immunofluorescence findings revealed decreased GFAP fluorescence intensity in the hippocampal CA1 and CA3 regions. This further confirmed that hippocampal neurons were damaged, suggesting that the model of cognitive dysfunction induced by MCAO\u0026thinsp;+\u0026thinsp;CRS was successfully established. Studies have indicated that depressed behavior in mice with PSD is mediated by IL-18 derived from neurons and microglia (Wu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been documented that H\u003csub\u003e2\u003c/sub\u003eS can counteract cognitive impairment caused by chronic stress in rats (Habibitabar et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). An increasing number of studies have demonstrated that H\u003csub\u003e2\u003c/sub\u003eS exerts significant neuroprotective effects on the nervous system by regulating neurogenesis (Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and autophagy (Kang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), antagonizing oxidative stress in nerve cells (Hu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and exerting anti-inflammatory effects (Wei et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, H\u003csub\u003e2\u003c/sub\u003eS improves chronic stress-induced pyroptosis in hippocampal cells, demonstrating its important neuroprotective effect. NaHS treatment suppressed the activation of GFAP, a specific astrocyte marker, in the hippocampus of PSD mice. Compared with the Sham\u0026thinsp;+\u0026thinsp;Vehicle group, the expressions of cleaved caspase-1, IL-18, and GFAP in the Sham\u0026thinsp;+\u0026thinsp;NaHS group did not change significantly. These results indicate that H\u003csub\u003e2\u003c/sub\u003eS ameliorates pyroptosis in the hippocampal cells of PSD mice. This study provides a new approach to prevent and treat PSD-related diseases. Next, we investigated whether H\u003csub\u003e2\u003c/sub\u003eS ameliorates hippocampal pyroptosis in PSD mice and whether it is associated with the level of related factors. Studies have revealed that H\u003csub\u003e2\u003c/sub\u003eS can improve chronic stress-induced pyroptosis in the hippocampus of rats by upregulating GDF11 expression during chronic stress (Ma et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). H\u003csub\u003e2\u003c/sub\u003eS has been found to improve spatial learning and memory in mice with Alzheimer's disease by inhibiting the activation of the NLRP3 inflammasome, preventing the inflammatory response, reducing neuronal damage, and avoiding the adverse effects of the continuous increase in inflammation in the body during chronic stress (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, previous studies by our research group have confirmed that the exogenous H\u003csub\u003e2\u003c/sub\u003eS donor, NaHS, can improve hippocampal damage caused by status epilepticus (Zhuang et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition to being highly susceptible to damage and stress responses, the hippocampus is a brain region that is closely related to behavior, depression, and mood. Because of these properties of the hippocampus, previous studies have explored changes in the hippocampal CA1 and CA3. Previous studies have suggested that activated astrocytes are involved in the pathophysiology of PSD. The present study identified decreased astrocytes and inflammatory factors, including cleaved caspase-1 and IL-18, in the CAl and CA3 of PSD mice after NaHS administration.\u003c/p\u003e \u003cp\u003eOur findings demonstrated that NaHS not only significantly alleviated depression-like behavior in PSD mice but also exhibited an excellent neuroprotective effect against ischemia/reperfusion injury. Nigericin activates NLRP3 inflammasome. Nigericin administration to NaHS-treated PSD mice revealed a significant increase in IL-18 and cleaved caspase-1 in the hippocampus of mice, indicating that nigericin reversed the protective effect of H\u003csub\u003e2\u003c/sub\u003eS. Therefore, NaHS alleviates cognitive dysfunction in PSD mice, and the mechanism may be related to the activation of astrocytes and the inhibition of inflammatory responses.\u003c/p\u003e \u003cp\u003ePSD seriously affects the physical and psychological rehabilitation and prognosis of stroke patients, and its incidence is increasing annually. There are many opinions on PSD at home and abroad, and prevention and treatment strategies are mainly focused on the use of antidepressants, such as serotonin reuptake inhibitors. Therefore, exploring the mechanisms and treatment of PSD may have long-term clinical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNo funding program supported this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSun wrote the main manuscript text and prepared figures 1-6. All authors reviewed the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData Availability Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experimental data are stored in the database of Hebei Provincial Key Laboratory of Integrated Traditional and Western Medicine in Neurological Rehabilitation, no open access web site, more raw data please refer to the supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eB\u0026eacute;langer M, Allaman I, Magistretti PJ (2011) Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metabol 14:724\u0026ndash;738\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuzs\u0026aacute;ki G, Draguhn A (2004) Neuronal oscillations in cortical networks. 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Neuroimmunomodulation \u003cem\u003e23\u003c/em\u003e, 271\u0026ndash;277\u003c/span\u003e\u003c/li\u003e\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":"","lastPublishedDoi":"10.21203/rs.3.rs-4557185/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4557185/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCognitive decline is common in post-stroke depression (PSD) and has been reported to be associated with oxidative stress. Hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS)\u0026mdash;an antioxidant gas molecule\u0026mdash;participates in producing the antioxidant glutathione, upregulating antioxidant signals, and maintaining mitochondrial integrity.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the neuroprotective effects of sodium hydrosulfide (NaHS, an H\u003csub\u003e2\u003c/sub\u003eS donor) on cognitive impairment after PSD. After subjecting middle cerebral artery occlusion (MCAO) mice to chronic restraint stress (CRS) for 21 days, NaHS 0.1 mmol/kg was injected intraperitoneally daily for seven consecutive days. The forced swimming test (FST) and fear conditioning test (FC) were used to evaluate depression-like behavior and cognitive function. NaHS administration significantly reversed MCAO plus CRS-induced PSD and cognitive impairment, including increased immobility time, reduced context-related freezing time, elevated astrocytic pyroptosis indicated by interleukin-18 and cleaved caspase-1 in the hippocampal CA1 and CA3, downregulated oscillations in theta, and upregulated oscillations in gamma under FC conditions. H\u003csub\u003e2\u003c/sub\u003eS provides a new perspective for treating cognitive impairment in PSD patients.\u003c/p\u003e","manuscriptTitle":"Exogenous hydrogen sulfide ameliorates memory dysfunction in post-stroke depressed mice by reducing NLRP3 inflammasome activation in astrocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-13 02:31:37","doi":"10.21203/rs.3.rs-4557185/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":"4f78ad1a-c2e5-4a6f-9e01-e59920f2a056","owner":[],"postedDate":"July 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-22T13:40:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-13 02:31:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4557185","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4557185","identity":"rs-4557185","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}