Intranasal dantrolene nanoparticles inhibit lipopolysaccharide-induced depression and anxiety behavior in mice

Intranasal dantrolene nanoparticles inhibit lipopolysaccharide-induced depression and anxiety behavior in mice | 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 Article Intranasal dantrolene nanoparticles inhibit lipopolysaccharide-induced depression and anxiety behavior in mice Huafeng Wei, Jia Liu, Yan Lu, Piplu Bhuiyan, Jacob Gruttner, Lauren St. Louis, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6254774/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Translational Psychiatry → Version 1 posted 12 You are reading this latest preprint version Abstract This study investigates the therapeutic effectiveness of intranasal dantrolene nanoparticles pretreatment to inhibit lipopolysaccharide (LPS)-induced pathological inflammation and synapse destruction and depressive and anxiety behavior in mice. B6SJLF1/J adult mice were pretreated with intranasal dantrolene nanoparticles (dantrolene: 5mg/kg), daily, Monday to Friday, 5 days per week, for 4 weeks. Then, mice were treated with an intraperitoneal injection of LPS (5mg/kg) for one time. Behavioral tests for depression and anxiety were performed 24 hours after a one-time LPS injection. Biomarkers for pyroptosis-related inflammation cytokines (IL-1β and IL-18) in the blood and brain were measured using enzyme-linked immunosorbent assay (ELISA) and immunoblotting, respectively. The changes of primary proteins activation inflammatory pyroptosis (NLRP3: NLR family pyrin domain containing 3, Caspase-1, N-GSDMD: N terminal protein gasdermin D) and synapse proteins (PSD-95 and synpatin-1) in brains were measured using immunoblotting. Intranasal dantrolene nanoparticles robustly inhibited LPS-induced depression and anxiety behavior. Intranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of IL-1β and IL-18 in the blood and brain and inhibited LPS-induced activation of pyroptosis. Intranasal dantrolene nanoparticles significantly ameliorated decrease of PSD-95 and synpatin-1 proteins in brains. Thus, intranasal dantrolene nanoparticles have demonstrated neuroprotection against inflammation-mediated depression and anxiety behaviors and should be studied further as a future effective drug treatment of major depression disorder or anxiety psychiatric disorder. Health sciences/Diseases/Psychiatric disorders/Depression Health sciences/Diseases/Psychiatric disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Three hundred million people worldwide suffer from major depressive disorder (MDD) 1 . MDD is a chronic and recurrent disease affecting about 20% of the population and the leading cause of suicide 2 . MDD imposes a tremendous psychological burden, as well as significant social repercussions and contributions to other disabilities 3 . The expected direct and indirect costs of MDD are up to $ 6 trillion in the USA alone 4 . An estimated 4% of the global population currently experience an anxiety disorder. In 2019, 301 million people in the world had an anxiety disorder, with a lifetime prevalence of approximately 34 percent 5 . Major depression disorder and anxiety disorders were approximately twice as prevalent among women 6 , 7 , with overall age-specific rates remaining relatively stable or increasing across the lifespan 8 . In 2021, an estimated 5.8 million (9.4%) children and adolescents were impacted by anxiety 9 . Depression and anxiety often co-exist and are the most common psychiatric diseases, which are chronic diseases with unclear mechanisms of pathology 10 . Classical treatment of depression or anxiety psychiatric disorders includes atypical antipsychotics that modulate dopamine and serotonin neurotransmission 11 , 12 . Pharmacotherapies for anxiety disorders also include drugs that interact with GABA neurotransmission systems 13 . Unfortunately, antidepressants have significant limitations, including slow onset of action, high rates of nonresponse, and acute worsening of anxiety 14 . Benzodiazepines are not recommended for long-term use in some anxiety disorders, due to concerns about their potential for abuse, tolerance, and withdrawal, and they are ineffective in some anxiety spectrum disorders 15 . Treatment of MDD is prone to a high risk of resistance (up to 30% of patients are unresponsive to the first treatment) and relapse (up to 8%) 3,10 . Treatment-resistant anxiety with remission rates may be as low as 25–35%, and relapse rates post remission may be 30% after 10 years 16 . Thus, there is an urgent need to develop novel approaches to treat depression and anxiety, especially treatment-resistant depression, or anxiety, with minimal side effects or organ toxicity. Increasing studies indicated that glutamate neurotransmission plays a critical role in mood function and its imbalance may cause psychiatric disorders 17 . Glutamate is linked to the development of anxiety disorders through its regulation of neuropeptides, fear extinction, and stress response. Glutamate is also important in synaptic and neural plasticity related to psychiatric disorders. Ketamine, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, was approved in 1970 by the Food and Drug Administration (FDA) for use in children and adults as an anesthetic. Recent studies indicate that ketamine is effective in treating MDD, especially treatment-resistant depression (TRD) 18 , 19 . Ketamine is also effective in treating anxiety disorders, even those resistant to traditional treatments 9 , 20 . While traditional antidepressants can take weeks to months to have an effect, ketamine has rapid effects on mood and suicidality, with mood changes reported as early as the first 4 hours after treatment 21 . As an antagonist of NMDAR, a primary glutamate receptor on the plasma membrane that causes Ca 2+ influx into the cytosol from extracellular space, ketamine has become a safe and broadly effective drug to treat depression or anxiety disorders, so is the intranasal esketamine 22 . This indicates that glutamate-mediated excitotoxicity and associated disruption of intracellular Ca 2+ homeostasis play a key role in the pathology of depression and anxiety disorders. Although molecular mechanisms are unclear, increasing evidence suggests that upstream disruption of intracellular Ca 2+ homeostasis and associated downstream inflammation and synapse dysfunction play critical roles in MDD pathologies 23 – 25 , as well as anxiety disorder 26 , 27 . The overactivation of ryanodine receptors (RyRs) in MDD and associated excessive Ca 2+ release from endoplasmic reticulum (ER) results in depletion of ER Ca 2+ and pathological elevation of cytosol and mitochondrial Ca 2+ concentrations, detrimental to synapse function and cell survival 25 , 28 , 29 . RyRs overactivation have also been shown to increase anxious behavior 28 . Upstream Ca 2+ dysregulation results in mitochondria dysfunction 30 , mitochondrial and cellular oxidative stress 10 , 31 , activation of inflammasomes, and cell or neuron death by pyroptosis 32 – 35 . This results in the release of inflammatory cytokines (IL-1β and IL-18) and pathological inflammation-mediated cell death by pyroptosis 34 , 36 . Pathological cytokines, especially pyroptosis-related IL-18 play important roles in psychiatric disorders 37 , 38 . Gut dysbiosis and associated inflammation also contribute to pathology in depression and anxiety behaviors 39 , 40 . Thus, a drug that inhibits upstream Ca 2+ dysregulation and then downstream pathological inflammation and programmed cell death by pyroptosis and synapse destruction is expected to treat MDD and anxiety disorder effectively. Dantrolene, a RyRs antagonist, is a US Food and Drug Administration-approved drug for the treatment of malignant hyperthermia, muscle spasms, and neuroleptic syndrome, with tolerable side effects and occasional liver toxicity at high doses 41 . Dantrolene, with its ability to inhibit the common upstream critical Ca 2+ dysregulation, is neuroprotective against many neurodegenerative diseases, including cerebral ischemia 42 , 43 , Huntington’s disease 44 , spinocerebellar ataxia 45 , amyotrophic lateral sclerosis 46 , and seizures 47 , 48 . Dantrolene robustly inhibited up to 78% of NMDA-induced elevation of cytosolic Ca 2+ concentration in cerebral cortical neurons 49 , suggesting it may have great potential to treat major depression disorder with a similar molecular mechanism of ketamine 50 , which needs to be studied urgently. In this study, we investigated the therapeutic effectiveness and the potential mechanisms of intranasal dantrolene nanoparticles on depression and anxiety behavior associated with pathological inflammation in adult mice. We induced inflammation by intraperitoneal injection of LPS. Our results demonstrated that intranasal dantrolene nanoparticles for twelve consecutive weeks robustly inhibited LPS-induced depression and anxiety behaviors in adult mice. These beneficial effects of intranasal dantrolene nanoparticles treatment were associated with its robust and significant inhibition of LPS-induced pathological elevation of pyroptosis-related inflammation cytokine of IL-1β and IL-18 in blood and brains and loss of synapse proteins (PSD-95 and synpatin-1) in brains. This study provides proof of concept that intranasal dantrolene nanoparticles may be an effective treatment for depression and anxiety psychiatric disorders, potentially by inhibiting pathological inflammation and synapse destruction in the CNS. Materials and methods Animals All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania. All B6SJLF1/J adult mice were obtained from Jackson Lab. Mice were divided into different cages according to age and gender, with no more than five mice per cage. Both male and female mice were used in this study. Experimental treatment groups As demonstrated in supplemental Fig. 1, adult male and female mice (5–10 months old) were randomly divided into different experimental groups. The mice were pretreated with either intranasal dantrolene nanoparticles (IN Dan, 5mg/kg) or vehicle (Veh), once a day, 5X/week (Monday through Friday). Control groups received no pretreatment. Fresh dantrolene nanoparticles were made every time before administration. Intranasal dantrolene nanoparticles stock solution was made at 5 mg/ml. The Ryanodex formulation vehicle was made fresh and contained all inactive ingredients in Ryanodex 51 , 52 . At the end of the 4-week pretreatment period, mice were treated with a one-time intraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg). Mice in the control group, without pretreatment, were treated with LPS (LPS, 5mg/kg) once as well. The sham control group received no pretreatment or treatment. At 24 hours following the one-time LPS treatment, behavioral tests for depression or anxiety behaviors were performed on all mice, with the following order of least to most stressful tests: OPT, EPMT, TST, and FST. Thereafter, all mice were euthanized, and the blood or brain was harvested for examination of inflammation and programmed cell death by pyroptosis and synapse destruction in the blood and brains. Intranasal dantrolene nanoparticle or vehicle administration Dantrolene (Sigma, St Louis, MO) was dissolved in the Ryanodex Formulation Vehicle (RFV: 125 mg mannitol, 25 mg polysorbate 80, 4mg povidone K12 in 5mL of sterile water and pH adjusted to 10.3), similar to our previous publications 52 , 53 . For intranasal administration, the final concentration of dantrolene was 5 mg/mL as we have previously described. Mice were held and fixed by the scruff of their necks with one hand. With the other hand, mice were given a total of 1µL/gram of body weight of dantrolene nanoparticles or RFV. Accordingly, a mouse weighing 20 g would be given 20µL of solution, equal to a dantrolene dose at 5mg/kg. The solution was slowly delivered directly into the mouse’s nose. Care was taken to ensure that mice were minimally stressed, and that the solution remained in the nasal cavity and did not enter the stomach or lungs. Forced swimming test (FST) Depression behavior was assessed using the FST, as described previously with some modification 54 . The mice from each group were settled in a clear glass tank of 25 cm height, 10 cm diameter, 15 cm water depth, and (23 ± 2) °C water temperature. The test's total period was 6 minutes, 2 minutes for adaptation, and the total immobility time was recorded in the next 4 minutes. Immobility occurred when the mice discontinued floundering on the surface of the water, instead appearing as floating in an equilibrium state. The greater the immobility time, the more severe the depression behavior. Tail suspension test (TST) Depression behavior was assessed using the tail suspension test described previously, with modifications 55 . We use specially manufactured tail suspension boxes, made of cartons with the dimensions (42 cm height X 18 cm width X 30 cm depth). To prevent animals from observing or interacting with each other, each mouse was suspended within its three-walled rectangular box. Each day, animals were acclimated to the testing room for at least 1 hour before the test. Each mouse was suspended in the middle of the box. The width and depth of the box were sufficiently sized to prevent the mouse from contacting the walls. In this setting, the approximate distance between the mouse's nose and the apparatus floor was 20–25 cm. A plastic suspension bar (50 cm. height X 40 cm. depth), was used to suspend the tail of each mouse and positioned on the top of the box. At the bottom of each box, we placed a paper towel to collect feces or urine from the animals. A dark grey box was used for albino animals and a white colored box for mice of other coat colors. Before evaluation, the tail of the mouse was securely adhered to the suspension bar, which was able to withstand the mouse’s weight. A video camera was placed in position and started recording the TST session. The total duration of the test was 6 minutes. The paper towel was replaced after each trial. After all sessions were finished, Any-maze software (Stoelting, USA) was used to analyze the collected data. During the behavioral analysis, the mobility time of each mouse was measured and subtracted from 360 seconds, producing the immobility time. The investigator was blinded to the animal groups. The higher immobility time, the more severe the depressive behavior. Open field test (OFT) The OFT was performed as previously described to evaluate anxiety behavior as described 56 . Each mouse was placed individually into the OFT apparatus (44 × 44 × 44 cm 3 ), facing the wall. The locomotor activity was recorded for six minutes with a camera above the apparatus under dim light. The anxiety behavior of each mouse was measured using the total distance traveled, center entries, immobility time, and time spent in the central zone. After each test, the apparatus was cleaned with 75% ethanol. Any-maze software (Stoelting USA) was used to analyze the collected data. The lesser the central zone distance and mean speed, the higher the anxiety behavior. The higher immobility time, the more severe the anxiety behavior. Elevated plus maze test (EPMT) Anxiety was also assessed using EPMT, as described previously, although with some modification 57 . Mice were placed in the center of an elevated plus-maze (arms are 33 cm × 5 cm, with 25 cm tall walls on the closed arms) under dim lighting and their behavior was videotaped for 5 minutes. We used Any-maze software (Stoelting USA) to analyze the collected data. The time spent in the closed and open arms, as well as the number of explorations of open arms, was measured and recorded. The more time spent in closed arms, the more severe the anxiety behavior. Euthanasia and tissue collection Mice were euthanized after the completion of all behavioral tests. As described previously 52 , mice were deeply anesthetized with 2–4% isoflurane delivered through a nose cone, with the concentration adjusted according to response to a toe pinch. The skin of each mouse was prepared, and an incision was made to open the chest and expose the heart. Blood was collected from the heart for the serum study using a syringe equipped with a 27G needle. The blood was centrifuged at 1400 rpm at 4◦C for 30 min, and the supernatant was collected and frozen at − 80◦C. The mice were euthanized by trans-cardial perfusion and exsanguination was conducted with cold phosphate-buffered saline. Measurements of serum concentration of IL-1β and IL-18 We measured the serum IL-1β and IL-18 cytokines using an ELISA kit, following company instructions, and as we described previously for measurement of S100β, with some modification 58 , 59 . All reagents and samples (the supernatant of the blood) were thawed to room temperature (18–25°C) before use. It is recommended that all standards and samples be run at least in duplicate. Add 100 µL of each standard and sample into appropriate wells. Cover wells and incubate for 2.5 hours at room temperature or overnight at 4°C with gentle shaking. Discard the solution and wash it 4 times with 1X Wash Solution. Wash by filling each well with Wash Buffer (300 µL) using a multi-channel Pipette or auto washer. Complete removal of liquid at each step is essential to reliable performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. Add 100 µL of 1x prepared Detection Antibody to each well. Cover wells and incubate for 1 hour at room temperature with gentle shaking. Discard the solution. Repeat the wash procedure as in step 3. Add 100 µL of prepared Streptavidin solution to each well. Cover wells and incubate for 45 minutes at room temperature with gentle shaking. Discard the solution. Repeat the wash as in step 3. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Cover wells and incubate for 30 minutes at room temperature in the dark with gentle shaking. Add 50 µL of Stop Solution (Item I) to each well. Read absorbance at 450 nm immediately. Mouse IL-1 beta ELISA Kit and Mouse IL18 ELISA Kit come from SIGMA(RAB0275-1KT/RAB08100-1KT). Immunoblotting (Western Blot) As we described previously 60 , total brain tissues were extracted by homogenization using cold RIPA buffer (#9806S, Cell Technology, USA) supplemented with protease inhibitor cocktails (P8340 Roche). The brain homogenates were rocked at 4°C for 90 minutes and then centrifuged at 14 000 rpm (Brushless Microcentrifuge, Denville 260D) for 20 minutes at 4°C to remove cell debris. After collecting the supernatant, the protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL 61101 USA). Briefly, equal amounts of protein (20µg/lane) were loaded onto 4–20% gel electrophoresis of mini-protein TGX precast (Cat. #4561094, BIO-RAD) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, MERK Millipore, Ireland) using wet electrotransfer system (BIO-RAD, USA). Following the transfer membrane blocking with 5% BSA (Sigma-Aldrich) for 1 hour, then the PVDF membrane was incubated with primary antibodies overnight at 4°C including NLRP3, cleaved caspase-1, GSDMD-NT, IL-1β, IL-18, PSD95, Synapsin-1, GAPDH (Table 1 ). Then, the membranes were incubated with a secondary antibody including anti-Mouse IgG1 HRP-linked; Anti-rabbit IgG, HRP-linked that was conjugated to horseradish peroxidase and washed with Tris-buffered saline containing 0.2% Tween-20 (TBST). Following that, TBST was used for washing the membranes three times for 10 minutes. After incubation with secondary antibodies, protein bands were visualized using ECL Western Blotting Detection Reagents (Cytiva, amersham, UK) and quantified for band intensity using ImageJ software (National Institutes of Health, Bethesda, MD, USA), by two persons blinded to treatments and averaged for each mouse. Table 1 Cat. and Manufacture Dilution Primary antibody Anti- NLRP3 68102-1-Ig; Proteintech 1:2 000 Anti-Human procaspase-1/ cleaved caspase-1 p20 #2225S, Cell signaling Technology,USA 1:1 000 Anti- Cleaved GSDMD #36425S; Cell signaling Technology, USA 1:1 000 Anti-IL-1β 31202S, Cell signaling Technology, USA 1:1 000 Anti-IL-18 10663-1-AP; Proteintech 1:3 000 Anti-PSD95 #2507; Cell signaling Technology, USA 1:1 000 Anti-Synapsin-1 #5297; Cell signaling Technology, USA 1:1 000 GAPDH MA5–15738, Thermo Fisher Scientific 1:10 000 Secondary antibody Anti-Mouse IgG1; HRP -linked # PA1-74421, Thermo Fisher Scientific 1:10 000 Anti-rabbit IgG, HRP-linked #7074; Cell signaling Technology, USA 1:1 000 Statistical Analysis All data were represented as mean ± standard deviations (Means ± SD). Statistical analyses were employed with GraphPad Prism (Version 9.3.1, CA, USA). Comparisons of more than two groups were conducted by one-way ANOVA with Tukey’s multiple comparison test. The N values in each group represent the number of mice. P < 0.05 was considered statistically significant. Results Intranasal dantrolene nanoparticle pretreatment significantly inhibited LPS-induced helplessness and anxiety-related behaviors. The FST and TST tests are commonly used to evaluate helplessness or depression behaviors in mice and to test the drug’s efficacy versus side effects 27 , 61 , 62 . In both FST (Fig. 1A) and TST (Fig. 1B), LPS induced significantly increased immobile times (helplessness or depression behaviors) by 89% (60.1 vs. 113.3) and 90% (98.1 vs. 186.6) respectively. Intranasal dantrolene nanoparticle pretreatment robustly inhibited the increased immobile time in FST by 41% (113.3 vs. 66.9) and in TST by 46% (186.6 vs. 101.5). In contrast, the intranasal dantrolene nanoparticles vehicle control did not demonstrate the same inhibitory effects on helplessness behaviors. The elevated plus maze test (EPMT) and open field test (OPT) are commonly used to evaluate anxiety-related behaviors in mice and test drug efficacy to treat anxiety behaviors 27 , 63 , 64 . In both EPMT (Fig. 1C) and OFT (Fig. 1D), LPS induced significantly increased time stay in closed arm in EPMT (anxiety behaviors) by 46% (162 vs. 237) and immobile time in OPF by 130% (82 vs. 189) respectively. Intranasal dantrolene nanoparticle pretreatment robustly inhibited the increased time stay in closed arm in EPMT by 29% (237 vs. 169) and immobile time in OPT by 40% (189 vs. 114). In contrast, the intranasal dantrolene nanoparticles vehicle control did not demonstrate the same inhibitory effects on depression behaviors. In the open field test, the less central zone distance mice traveled and had a lower moving speed, the more severe the anxiety behavior (supplemental Fig. 2). Compared to the control without treatment, one-time LPS treatment significantly decreased mean speed (anxiety behavior) but not traveled zone distance. Intranasal dantrolene nanoparticles tended to inhibit LPS-induced decrease of travel zone distance and mean speed (Supplemental Fig. 2). Intranasal dantrolene nanoparticle pretreatment significantly inhibited LPS-induced helplessness and anxiety behaviors primarily in female mice. We further analyze the effects of sex on neuroprotective effects of intranasal dantrolene nanoparticles on helplessness and anxiety behavior. As demonstrated in Fig. 2, intranasal dantrolene nanoparticles but not its vehicle control primarily inhibited helpless behavior determined by both FST (Fig. 2A) and TST (Fig. 2B) in female but not male mice. For anxiety behavior, although intranasal dantrolene nanoparticle but not vehicle control inhibited LPS-induced anxiety behavior determined by EPMT (Fig. 2C) in male mice, the inhibitory effects of dantrolene on anxiety behaviors were most significant in female mice determined by both EPMT (Fig. 2C) and OFT (Fig. 2D). Intranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of pyroptosis-related inflammation cytokines in blood and brains. Compared to control without treatment, IP injection of LPS (5mg/kg) for one time significantly increased blood concentrations of IL-1β by 147% (Fig. 3A, 136 vs. 336) and IL-18 by 67% (Fig. 3B, 233 vs. 390). Intranasal dantrolene nanoparticles but not its vehicle control significantly inhibited the increased IL-1β by 46% (336 vs. 183) and elevated IL-18 by 23% (Fig. 3B, 390 vs. 299). Furthermore, intranasal dantrolene nanoparticles significantly inhibited LPS-induced elevation of IL-1β and IL-18 proteins in both blood (Fig. 3A, B) and in brains (Fig. 3C, D, supplemental Figs. 3 and 4). Intranasal dantrolene nanoparticles inhibited LPS-induced programmed cell death by pyroptosis in mice brains. Compared to control mice without treatment, LPS treatment increased the protein levels of pyroptosis pathway activation (NLRP3, caspase-1 and N terminal GSDMD) in mice brains. Intranasal dantrolene nanoparticles significantly inhibited increase of Caspase-1 (P20), a biomarker of programmed cell death by pyroptosis (Fig. 4C, D, supplemental Fig. 6), and trended to inhibit LPS-induced elevation of pyroptosis pathway activation proteins (Fig. 4, A and B, supplemental Fig. 5, NLRP-3 and Fig. 4E and F, supplemental Fig. 7, N terminal GSDMD (GSDMD-NT)) Intranasal dantrolene nanoparticles significantly inhibited LPS-induced synapse protein loss in the mice brains. Compared to control without treatment, LPS significantly decreased the synapse proteins of PSD-95 by 45% (1.322 vs. 0.726, Fig. 5, A, C, supplemental Fig. 8) and synapsin-1 by 37% (0.925 vs. 0.579, Fig. 5, B, D, supplemental Fig. 9) in mice brains. Intranasal dantrolene nanoparticles but not vehicle control pretreatment for 4 weeks significantly inhibited the pathologically reduction of synapse proteins of PSD-95 by 77% (0.726 vs. 1.286, Fig. 5A, C, supplemental Fig. 8) and synpasin-1 by 76% (0.579 vs. 1.021, Fig. 5B and D, supplemental Fig. 9). Discussion There is an urgent need for new therapeutic interventions to prevent or treat MDD 3 , 65 , and anxiety 5 psychiatric disorders, especially those drug-resistant and recurrent depression and anxiety 5 , 16 , 66 . This study has demonstrated that intranasal dantrolene nanoparticles are capable of ameliorating helplessness and anxiety behaviors in adult mice, associated with its ability to inhibit pathological elevation of pyroptosis related cytokines (IL-1β and IL-18) in blood, pyroptosis biomarker caspase-1 (P20) and synapse proteins loss in brains. Considering that upstream Ca 2+ dysregulation plays an important role in the pathology of depression and anxiety, evidenced by the effectiveness of ketamine and esketamine in treating depression and anxiety resistant to traditional drug treatments 14 , 16 , a new class of drugs, such as dantrolene, could be developed with a mechanism to correct the disruption of upstream Ca 2+ dysregulation and associated downstream pathological inflammation and pyroptosis in psychiatric disorders. Increasing studies suggest that inflammation is an important pathology causing MDD and anxiety and could be a target for effective treatments 67 – 70 . Intracellular Ca 2+ dysregulation has been proposed as the upstream cause of downstream mitochondria dysfunction, oxidative stress, and inflammation in MDD 25 , 71 , 72 and anxiety 38 , 73 . Inhibition of upstream Ca 2+ dysregulation through inhibiting over-activation of RyRs has been shown to suppress inflammation in Gulf War illness characterized by depression and dementia 28 . Although dantrolene has been shown to inhibit pathological inflammation with increased cytokines in diabetes 74 , 75 , sepsis 76 , 77 , and COVID-19 infection 78 , 79 , its effects to inhibit Ca 2+ dysregulation and associated inflammation for treating depression and anxiety behaviors have not yet been investigated until now and studied herewith. Several animal models to establish depression and anxiety behaviors have been suggested based on the proposed neurobiology mechanisms, including altered neurotransmission, HPA axis abnormalities involved in chronic stress, inflammation, reduced neuroplasticity, and network dysfunction 80 – 82 . Induction of inflammation in animals has been increasingly used to establish depression and anxiety animal models, especially using LPS to induce inflammation, depression, and anxiety behaviors to test drug treatment efficacy 37 , 71 , 83 – 86 . The common approach of LPS administration is intraperitoneal injection because of its ease and feasibility. The dose and duration of IP injection of LPS to establish depression or anxiety behaviors has been varied but appears to be dose dependent. The high dose of LPS at 5 mg/kg, administered once, seems to be dependable in inducing inflammation and establishing depression behavior 71 . Our study confirmed the efficiency of a one-time administration of IP LPS (5 mg/kg) to induce inflammation and depression and anxiety behavior in adult mice consistently. Accordingly, the LPS-induced inflammation-mediated depression behaviors are adequate to evaluate the therapeutic effects of dantrolene to treat depression. Our results demonstrated robust inhibition of LPS-induced inflammation and associated helplessness and anxiety behaviors by intranasal dantrolene nanoparticles pretreatment in adult mice. In addition to the protective effects of dantrolene against memory loss in various AD animal models 52 , 87 – 89 , intranasal dantrolene nanoparticles also significantly inhibited helplessness and anxiety behaviors, and associated inflammation and pyroptosis, suggesting that pathological inflammation and pyroptosis play a critical role in both cognitive dysfunction and psychiatric disorders. Drugs that target inflammation pathology should be developed to treat both memory loss and psychiatric disorders in AD patients. Nevertheless, intranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of pyroptosis related inflammation cytokines (IL-1β and IL-18) in blood and ameliorated both depression and anxiety behavior in adult mice, suggesting that intranasal dantrolene nanoparticles could be developed as an effective drug to treat depression and/or anxiety in major depression disorder (MDD) patients potentially by its inhibition of inflammation and associated programmed cell death by pyroptosis. MDD affects female patients twice as much as male 90 , 91 . The proposed mechanisms for this sex difference include the effects of estrogen 92 , serotonin, and tryptophan metabolism 93 , different stress reactions 91 , etc. An important finding in this study is that intranasal dantrolene nanoparticles are more effective in ameliorating helplessness and anxiety behaviors in female than in male mice. Our result is consistent with MDD being more prevalent in female mice than in male. Also, the more significant inhibition of inflammation associated with helplessness and anxiety behavior in female mice by intranasal dantrolene nanoparticles suggested that dantrolene may inhibit critical pathology and etiology contributing to depression and anxiety behaviors. Although the exact mechanism is unclear, we propose that dantrolene inhibited the upper stream critical Ca 2+ dysregulation and associated inflammation and associated programmed neuron death by pyroptosis and synapse protein loss. Intranasal administration of drugs, especially in a nanoparticle formulation, significantly promotes drugs that bypass the blood-brain barrier (BBB) and penetrate the CNS, with reduced peripheral toxicity 94 – 96 . Our recent research work indicated that intranasal dantrolene nanoparticles achieved higher therapeutic efficacy in the brain compared to oral and subcutaneous forms of administration, with minimal or no side effects after chronic use 52 , 53 , 97 . Intranasal dantrolene nanoparticles significantly ameliorated memory loss in adult 5XFAD mice as a disease-modifying drug 52 . Increasing studies suggest ryanodine receptor overactivation and associated Ca 2+ dysregulation is an upstream critical pathology leading to multiple downstream pathologies including mitochondria dysfunction 98 , oxidative stresses 99 , pathological inflammation 100 , and neuron damage by pyroptosis 101 . Eventually, these pathologies result in depression and/or anxiety psychiatric disorders 34 , 70 , 102 . This study demonstrated that inhibition of RyRs overactivation by dantrolene inhibited pathological inflammation, synapse proteins destruction, and ameliorated depression and anxiety behavior robustly. This study further strengthens the indication that upstream RyRs overactivation and Ca 2+ dysregulation and associated downstream pathological inflammation and synapse destruction play important roles in depression and anxiety psychiatric disorders. Like esketamine 22 , 103 , the intranasal dantrolene nanoparticles could become an effective disease-modifying drug treatment for both depression and anxiety psychiatric disorders and need to be investigated in future clinical studies. Since depression and anxiety psychiatric disorders are chronic diseases, they require long-term drug treatments. Accordingly, proposed drugs must limit side effects or organ toxicity with chronic treatment. The major advantage of using intranasal dantrolene nanoparticles, in comparison to commonly used oral or intravenous approaches, is that it significantly increases the brain/blood concentration ratio of dantrolene 52 , 53 , especially in aged mice 97 . This promotes its CNS therapeutic effects, while minimizing its side effects or organ toxicity 52 . Our previous study demonstrated no side effects on nose structure and smell function 52 , 104 , liver structure 52 and function, or muscle function 51 , 52 , 87 after up to 10 months of chronic treatment in adult 5XFAD mice 52 , suggesting that intranasal dantrolene nanoparticles are safe to be used chronically in animals. Chronic use of dantrolene in human patients needs to be investigated further. This study has the following limitations: 1. The mice are from 5 to 10 months old. Although they are considered adult mice, future studies need to focus on different age groups, especially in aged mice, considering the high prevalence of psychiatric disorders in the aged population. 2). We were unable to measure the changes of cytosol versus mitochondria Ca 2+ levels in the brain tissue due to technological challenges. However, dantrolene has been shown to inhibit LPS or AD gene mutation-induced overactivation of RyRs and associated Ca 2+ dysregulation in different cell culture models 105 , 106 . 3). We did not measure the contents of reactive oxygen species (ROS) concentrations in the brain, which is usually the upstream pathology to activate NLRP3 inflammasome 107 . 4). We did not measure other biomarkers demonstrating effects of intranasal dantrolene nanoparticles on programmed cell death by pyroptosis in brains. 6). We did not study the dose-response of dantrolene to treat depression and anxiety behaviors. Physiological Ca 2+ release from ER is important for many physiological functions in cells, which means over-inhibition of calcium release from the ER may be harmful to cells, as we described previously. The adequate dose of intranasal dantrolene nanoparticles to treat psychiatric disorders must be investigated thoroughly before its clinical use in patients. In conclusion, this study demonstrated that intranasal dantrolene nanoparticles significantly ameliorated inflammation-mediated depression and anxiety behaviors in adult mice. This neuroprotective effect against depression and anxiety behaviors was associated with its robust inhibition of pathological elevation of pyroptosis-related inflammation cytokines in blood and the synapse destruction in the brains. This study may inspire future studies to repurpose intranasal dantrolene nanoparticles in treating depression and anxiety psychiatric disorders. Declarations Conflict of Interests Drs. Huafeng Wei and Ge Liang are listed as inventors of patent applications entitled “Intranasal Administration of Dantrolene for Treatment of Alzheimer’s Disease” in multiple countries by The Trustees of the University of Pennsylvania. Author contributions H.W. conceived and designed the study. J.L., Y.L, P.B. J.G, L.S.L, Y.Y., G.L, H.W. conducted experiments, acquired and analyzed the data, J.L., Y.L, P.B. J.G, L.S.L, Y.Y., G.L, H.W. analyzed data and contributed to the manuscript preparation. H.W. wrote the manuscript. All the authors reviewed and approved the final manuscript. Acknowledgments This work was supported by grants to HW from the National Institute on Aging (R01AG061447) and NIA R01 Supplemental (3R01AG061447-03S1). The research was performed in the lab of Dr. Huafeng Wei and should be attributed to the Department of Anesthesiology, University of Pennsylvania. We appreciate the technical support from Rebecca Chae from Rowan University, New Jersey, U.S.A. and Kyulee Kim from UPENN, Pennsylvania, U.S.A. References Herrman H, Kieling C, McGorry P, Horton R, Sargent J, Patel V: Reducing the global burden of depression: a Lancet-World Psychiatric Association Commission. Lancet 2019; 393: e42-e43 Rikhani K, Vas C, Jha MK: Approach to Diagnosis and Management of Treatment-Resistant Depression. Psychiatr Clin North Am 2023; 46: 247–259 Belge JB, Sabbe ACF, Sabbe B: An update on pharmacotherapy for recurrent depression in 2022. Expert Opin Pharmacother 2023: 1–8 Gururajan A, Reif A, Cryan JF, Slattery DA: The future of rodent models in depression research. Nat Rev Neurosci 2019; 20: 686–701 Szuhany KL, Simon NM: Anxiety Disorders: A Review. JAMA 2022; 328: 2431–2445 Jarkas DA, Villeneuve AH, Daneshmend AZB, Villeneuve PJ, McQuaid RJ: Sex differences in the inflammation-depression link: A systematic review and meta-analysis. Brain Behav Immun 2024; 121: 257–268 Altemus M, Sarvaiya N, Neill Epperson C: Sex differences in anxiety and depression clinical perspectives. Front Neuroendocrinol 2014; 35: 320–30 Somers JM, Goldner EM, Waraich P, Hsu L: Prevalence and incidence studies of anxiety disorders: a systematic review of the literature. Can J Psychiatry 2006; 51: 100–13 Bruton AM, Wesemann DG, Machingo TA, Majak G, Johnstone JM, Marshall RD: Ketamine for mood disorders, anxiety, and suicidality in children and adolescents: a systematic review. Eur Child Adolesc Psychiatry 2024 Ait Tayeb AEK, Poinsignon V, Chappell K, Bouligand J, Becquemont L, Verstuyft C: Major Depressive Disorder and Oxidative Stress: A Review of Peripheral and Genetic Biomarkers According to Clinical Characteristics and Disease Stages. Antioxidants (Basel) 2023; 12 Cowen PJ: SSRIs in the Treatment of Depression: A Pharmacological CUL-DE-SAC? Curr Top Behav Neurosci 2024; 66: 1–19 Malena K, Rao S, Mosler C: Review of Current Clinical Options for the Management of Behavioral and Psychological Symptoms of Dementia. Sr Care Pharm 2024; 39: 300–310 Nuss P, Ferreri F, Bourin M: An update on the anxiolytic and neuroprotective properties of etifoxine: from brain GABA modulation to a whole-body mode of action. Neuropsychiatr Dis Treat 2019; 15: 1781–1795 Paganin W, Signorini S, Leccese V, Sciarretta A: [Treatment-resistant depression. From classification to new therapies.]. Riv Psichiatr 2022; 57: 258–272 Robertson S, Peacock EE, Scott R: Benzodiazepine Use Disorder: Common Questions and Answers. Am Fam Physician 2023; 108: 260–266 Tabaac BJ, Shinozuka K, Arenas A, Beutler BD, Cherian K, Evans VD et al: Psychedelic Therapy: A Primer for Primary Care Clinicians-Historical Perspective and Overview. Am J Ther 2024; 31: e97-e103 Sarawagi A, Soni ND, Patel AB: Glutamate and GABA Homeostasis and Neurometabolism in Major Depressive Disorder. Front Psychiatry 2021; 12: 637863 Ren L: The mechanistic basis for the rapid antidepressant-like effects of ketamine: From neural circuits to molecular pathways. Prog Neuropsychopharmacol Biol Psychiatry 2024; 129: 110910 Costi S: Ketamine for Major Depressive Disorder. Curr Top Behav Neurosci 2023 Tully JL, Dahlen AD, Haggarty CJ, Schioth HB, Brooks S: Ketamine treatment for refractory anxiety: A systematic review. Br J Clin Pharmacol 2022; 88: 4412–4426 Matveychuk D, Thomas RK, Swainson J, Khullar A, MacKay MA, Baker GB et al: Ketamine as an antidepressant: overview of its mechanisms of action and potential predictive biomarkers. Ther Adv Psychopharmacol 2020; 10: 2045125320916657 Rosso G, d'Andrea G, Barlati S, Di Nicola M, Andriola I, Marcatili M et al: Esketamine Treatment Trajectory of Patients with Treatment-Resistant Depression in the Mid and Long-Term Run: Data from REAL-ESK Study Group. Curr Neuropharmacol 2025; 23: e1570159X337670 Deutschenbaur L, Beck J, Kiyhankhadiv A, Muhlhauser M, Borgwardt S, Walter M et al: Role of calcium, glutamate and NMDA in major depression and therapeutic application. Prog Neuropsychopharmacol Biol Psychiatry 2016; 64: 325–33 Fries GR, Saldana VA, Finnstein J, Rein T: Molecular pathways of major depressive disorder converge on the synapse. Mol Psychiatry 2023; 28: 284–297 Garrosa-Jimenez J, Sanchez Carro Y, Ovejero-Benito MC, Del Sastre E, Garcia AG, Lopez MG et al: Intracellular calcium and inflammatory markers, mediated by purinergic stimulation, are differentially regulated in monocytes of patients with major depressive disorder. Neurosci Lett 2021; 765: 136275 Valiati FE, Feiten JG, Gea LP, Silveira EMJ, Scotton E, Caldieraro MA et al: Inflammation and damage-associated molecular patterns in major psychiatric disorders. Trends Psychiatry Psychother 2022 Wang Y, Wu LH, Hou F, Wang ZJ, Wu MN, Holscher C et al: Mitochondrial calcium uniporter knockdown in hippocampal neurons alleviates anxious and depressive behavior in the 3XTG Alzheimer's disease mouse model. Brain Res 2024; 1840: 149060 Phillips KF, Santos E, Blair RE, Deshpande LS: Targeting Intracellular Calcium Stores Alleviates Neurological Morbidities in a DFP-Based Rat Model of Gulf War Illness. Toxicol Sci 2019; 169: 567–578 Cardon I, Grobecker S, Kucukoktay S, Bader S, Jahner T, Nothdurfter C et al: Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease. Int J Mol Sci 2024; 25 Song Y, Cao H, Zuo C, Gu Z, Huang Y, Miao J et al: Mitochondrial dysfunction: A fatal blow in depression. Biomed Pharmacother 2023; 167: 115652 Lindqvist D, Dhabhar FS, James SJ, Hough CM, Jain FA, Bersani FS et al: Oxidative stress, inflammation and treatment response in major depression. Psychoneuroendocrinology 2017; 76: 197–205 Zhou XY, Thai M, Roediger D, Mueller BA, Cullen KR, Klimes-Dougan B et al: Mitochondrial health, NLRP3 inflammasome activation, and white matter integrity in adolescent mood disorders: A pilot study. J Affect Disord 2023; 340: 149–159 Cao H, Yang D, Nie K, Lin R, Peng L, Zhou X et al: Hesperidin may improve depressive symptoms by binding NLRP3 and influencing the pyroptosis pathway in a rat model. Eur J Pharmacol 2023; 952: 175670 Li S, Sun Y, Song M, Song Y, Fang Y, Zhang Q et al: NLRP3/caspase-1/GSDMD-mediated pyroptosis exerts a crucial role in astrocyte pathological injury in mouse model of depression. JCI Insight 2021; 6 Simon MS, Schiweck C, Arteaga-Henriquez G, Poletti S, Haarman BCM, Dik WA et al: Monocyte mitochondrial dysfunction, inflammaging, and inflammatory pyroptosis in major depression. Prog Neuropsychopharmacol Biol Psychiatry 2021; 111: 110391 Kaufmann FN, Costa AP, Ghisleni G, Diaz AP, Rodrigues ALS, Peluffo H et al: NLRP3 inflammasome-driven pathways in depression: Clinical and preclinical findings. Brain Behav Immun 2017; 64: 367–383 Vogelzangs N, de Jonge P, Smit JH, Bahn S, Penninx BW: Cytokine production capacity in depression and anxiety. Transl Psychiatry 2016; 6: e825 Zhang LM, Zhang DX, Song RX, Lv JM, Wang LY, Wu ZY et al: IL-18BP Alleviates Anxiety-Like Behavior Induced by Traumatic Stress via Inhibition of the IL-18R-NLRP3 Signaling Pathway in a Mouse Model of Hemorrhagic Shock and Resuscitation. Mol Neurobiol 2023; 60: 382–394 Bhatt S, Kanoujia J, Mohana Lakshmi S, Patil CR, Gupta G, Chellappan DK et al: Role of Brain-Gut-Microbiota Axis in Depression: Emerging Therapeutic Avenues. CNS Neurol Disord Drug Targets 2023; 22: 276–288 Anand N, Gorantla VR, Chidambaram SB: The Role of Gut Dysbiosis in the Pathophysiology of Neuropsychiatric Disorders. Cells 2022; 12 Ratto D, Joyner RW: Dantrolene, StatPearls. Treasure Island (FL), 2020 Nakayama R, Yano T, Ushijima K, Abe E, Terasaki H: Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CA1 region subjected to transient ischemia. Anesthesiology 2002; 96: 705–10 Wei HF, Perry DC: Dantrolene is cytoprotective in two models of neuronal cell death. Journal of Neurochemistry 1996; 67: 2390–2398 Chen X, Wu J, Lvovskaya S, Herndon E, Supnet C, Bezprozvanny I: Dantrolene is neuroprotective in Huntington's disease transgenic mouse model. Mol Neurodegener 2011; 6: 81 Chen X, Tang TS, Tu H, Nelson O, Pook M, Hammer R et al: Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3. J Neurosci 2008; 28: 12713–24 Rothstein JD, Kuncl RW: Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity. J Neurochem 1995; 65: 643–51 Royero PX, Higa GSV, Kostecki DS, Dos Santos BA, Almeida C, Andrade KA et al: Ryanodine receptors drive neuronal loss and regulate synaptic proteins during epileptogenesis. Exp Neurol 2020; 327: 113213 Keshavarz M, Fotouhi M, Rasti A: Dantrolene: A Selective Ryanodine Receptor Antagonist, Protects Against Pentylenetetrazole-Induced Seizure in Mice. Acta Med Iran 2016; 54: 555–561 Frandsen A, Schousboe A: Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons. Proc Natl Acad Sci U S A. 1992; 89: 2590–2594 Chen T, Cheng L, Ma J, Yuan J, Pi C, Xiong L et al: Molecular mechanisms of rapid-acting antidepressants: New perspectives for developing antidepressants. Pharmacol Res 2023; 194: 106837 Vera R, Hong N, Jiang B, Liang G, Eckenhoff MF, Kincaid HJ et al: Effects of intranasal dantrolene nanoparticles on brain concentration and behavior in PS19 tau transgenic mice. Res Sq 2023 Shi Y, Zhang L, Gao X, Zhang J, Ben Abou M, Liang G et al: Intranasal Dantrolene as a Disease-Modifying Drug in Alzheimer 5XFAD Mice. J Alzheimers Dis 2020; 76: 1375–1389 Wang J, Shi Y, Yu S, Wang Y, Meng Q, Liang G et al: Intranasal administration of dantrolene increased brain concentration and duration. PLoS One 2020; 15: e0229156 Ueno H, Takahashi Y, Murakami S, Wani K, Matsumoto Y, Okamoto M et al: Effect of simultaneous testing of two mice in the tail suspension test and forced swim test. Sci Rep 2022; 12: 9224 Can A, Dao DT, Terrillion CE, Piantadosi SC, Bhat S, Gould TD: The tail suspension test. J Vis Exp 2012: e3769 Jones SK, McCarthy DM, Vied C, Stanwood GD, Schatschneider C, Bhide PG: Transgenerational transmission of aspartame-induced anxiety and changes in glutamate-GABA signaling and gene expression in the amygdala. Proc Natl Acad Sci U S A 2022; 119: e2213120119 Cho WH, Noh K, Lee BH, Barcelon E, Jun SB, Park HY et al: Hippocampal astrocytes modulate anxiety-like behavior. Nat Commun 2022; 13: 6536 Joseph DJ, Liu C, Peng J, Liang G, Wei H: Isoflurane mediated neuropathological and cognitive impairments in the triple transgenic Alzheimer's mouse model are associated with hippocampal synaptic deficits in an age-dependent manner. PLoS One 2019; 14: e0223509 Liang G, Ward C, Peng J, Zhao YF, Huang BS, Wei HF: Isoflurane Causes Greater Neurodegeneration Than an Equivalent Exposure of Sevoflurane in the Developing Brain of Neonatal Mice. Anesthesiology 2010; 112: 1325–1334 Wang Y, Liang G, Liang S, Mund R, Shi Y, Wei H: Dantrolene Ameliorates Impaired Neurogenesis and Synaptogenesis in Induced Pluripotent Stem Cell Lines Derived from Patients with Alzheimer's Disease. Anesthesiology 2020; 132: 1062–1079 Liu QF, Choi H, Son T, Kim YM, Kanmani S, Chin YW et al: Co-Treatment with the Herbal Medicine SIP3 and Donepezil Improves Memory and Depression in the Mouse Model of Alzheimer's Disease. Curr Alzheimer Res 2022; 19: 246–263 Amada N, Hirose T, Suzuki M, Kakumoto Y, Futamura T, Maeda K et al: Synergistic anti-depressive effect of combination treatment of Brexpiprazole and selective serotonin reuptake inhibitors on forced swimming test in mice. Neuropsychopharmacol Rep 2023; 43: 132–136 Varkonyi D, Torok B, Sipos E, Fazekas CL, Banrevi K, Correia P et al: Investigation of Anxiety- and Depressive-like Symptoms in 4- and 8-Month-Old Male Triple Transgenic Mouse Models of Alzheimer's Disease. Int J Mol Sci 2022; 23 Pentkowski NS, Rogge-Obando KK, Donaldson TN, Bouquin SJ, Clark BJ: Anxiety and Alzheimer's disease: Behavioral analysis and neural basis in rodent models of Alzheimer's-related neuropathology. Neurosci Biobehav Rev 2021; 127: 647–658 Brady LS, Lisanby SH, Gordon JA: New directions in psychiatric drug development: promising therapeutics in the pipeline. Expert Opin Drug Discov 2023: 1–16 Jha MK, Mathew SJ: Pharmacotherapies for Treatment-Resistant Depression: How Antipsychotics Fit in the Rapidly Evolving Therapeutic Landscape. Am J Psychiatry 2023; 180: 190–199 Han Q, Li W, Chen P, Wang L, Bao X, Huang R et al: Microglial NLRP3 inflammasome-mediated neuroinflammation and therapeutic strategies in depression. Neural Regen Res 2024; 19: 1890–1898 Su WJ, Hu T, Jiang CL: Cool the Inflamed Brain: A Novel Anti-inflammatory Strategy for the Treatment of Major Depressive Disorder. Curr Neuropharmacol 2024; 22: 810–842 Chin Fatt CR, Mayes TL, Trivedi MH: Immune Dysregulation in Treatment-Resistant Depression: Precision Approaches to Treatment Selection and Development of Novel Treatments. Psychiatr Clin North Am 2023; 46: 403–413 Guo B, Zhang M, Hao W, Wang Y, Zhang T, Liu C: Neuroinflammation mechanisms of neuromodulation therapies for anxiety and depression. Transl Psychiatry 2023; 13: 5 Arioz BI, Tastan B, Tarakcioglu E, Tufekci KU, Olcum M, Ersoy N et al: Melatonin Attenuates LPS-Induced Acute Depressive-Like Behaviors and Microglial NLRP3 Inflammasome Activation Through the SIRT1/Nrf2 Pathway. Front Immunol 2019; 10: 1511 Resende R, Fernandes T, Pereira AC, De Pascale J, Marques AP, Oliveira P et al: Mitochondria, endoplasmic reticulum and innate immune dysfunction in mood disorders: Do Mitochondria-Associated Membranes (MAMs) play a role? Biochim Biophys Acta Mol Basis Dis 2020; 1866: 165752 Zhou GQ, Wang X, Gao P, Qin TZ, Guo L, Zhang ZW et al: Intestinal microbiota via NLRP3 inflammasome dependent neuronal pyroptosis mediates anxiety-like behaviour in mice exposed to 3.5 GHz radiofrequency radiation. Sci Total Environ 2024; 927: 172391 Clark AL, Kanekura K, Lavagnino Z, Spears LD, Abreu D, Mahadevan J et al: Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death. Sci Rep 2017; 7: 5611 Johnson JD, Han Z, Otani K, Ye H, Zhang Y, Wu H et al: RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets. J Biol Chem 2004; 279: 24794–802 Fischer DR, Sun X, Williams AB, Gang G, Pritts TA, James JH et al: Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats. Shock 2001; 15: 200–7 Hotchkiss RS, Karl IE: Dantrolene ameliorates the metabolic hallmarks of sepsis in rats and improves survival in a mouse model of endotoxemia. Proc Natl Acad Sci U S A. 1994; 91: 3039–3043 Al-Kuraishy HM, Al-Gareeb AI, El-Bouseary MM, Alexiou A, Papadakis M, Batiha GE: Calpain/calpstatin pathway in COVID-19: Role of dantrolene. J Cell Mol Med 2024; 28: e18005 Alkazmi L, Al-Kuraishy HM, Al-Gareeb AI, El-Bouseary MM, Ahmed EA, Batiha GE: Dantrolene and ryanodine receptors in COVID-19:The daunting taskand neglected warden. Clin Exp Pharmacol Physiol 2023 Villas Boas GR, Boerngen de Lacerda R, Paes MM, Gubert P, Almeida W, Rescia VC et al: Molecular aspects of depression: A review from neurobiology to treatment. Eur J Pharmacol 2019; 851: 99–121 Dean J, Keshavan M: The neurobiology of depression: An integrated view. Asian J Psychiatr 2017; 27: 101–111 Sapolsky RM: Psychiatric distress in animals versus animal models of psychiatric distress. Nat Neurosci 2016; 19: 1387–1389 Mahdirejei HA, Peeri M, Azarbayjani MA, Fattahi Masrour F: Fluoxetine combined with swimming exercise synergistically reduces lipopolysaccharide-induced depressive-like behavior by normalizing the HPA axis and brain inflammation in mice. Pharmacol Biochem Behav 2023; 232: 173640 Zhuo R, Cheng X, Luo L, Yang L, Zhao Y, Zhou Y et al: Cinnamic Acid Improved Lipopolysaccharide-Induced Depressive-Like Behaviors by Inhibiting Neuroinflammation and Oxidative Stress in Mice. Pharmacology 2022; 107: 281–289 Li W, Ali T, He K, Liu Z, Shah FA, Ren Q et al: Ibrutinib alleviates LPS-induced neuroinflammation and synaptic defects in a mouse model of depression. Brain Behav Immun 2021; 92: 10–24 Li W, Xu Y, Liu Z, Shi M, Zhang Y, Deng Y et al: TRPV4 inhibitor HC067047 produces antidepressant-like effect in LPS-induced depression mouse model. Neuropharmacology 2021; 201: 108834 Peng J, Liang G, Inan S, Wu Z, Joseph DJ, Meng Q et al: Dantrolene ameliorates cognitive decline and neuropathology in Alzheimer triple transgenic mice. Neurosci Lett 2012; 516: 274–9 Chakroborty S, Briggs C, Miller MB, Goussakov I, Schneider C, Kim J et al: Stabilizing ER Ca2 + channel function as an early preventative strategy for Alzheimer's disease. PLoS One 2012; 7: e52056 Oules B, Del Prete D, Greco B, Zhang X, Lauritzen I, Sevalle J et al: Ryanodine receptor blockade reduces amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer disease. J Neurosci 2012; 32: 11820–34 Shors TJ, Millon EM, Chang HY, Olson RL, Alderman BL: Do sex differences in rumination explain sex differences in depression? J Neurosci Res 2017; 95: 711–718 Fernandez-Guasti A, Fiedler JL, Herrera L, Handa RJ: Sex, stress, and mood disorders: at the intersection of adrenal and gonadal hormones. Horm Metab Res 2012; 44: 607–18 Iqbal J, Huang GD, Xue YX, Yang M, Jia XJ: Role of estrogen in sex differences in memory, emotion and neuropsychiatric disorders. Mol Biol Rep 2024; 51: 415 Songtachalert T, Roomruangwong C, Carvalho AF, Bourin M, Maes M: Anxiety Disorders: Sex Differences in Serotonin and Tryptophan Metabolism. Curr Top Med Chem 2018; 18: 1704–1715 Maher R, Moreno-Borrallo A, Jindal D, Mai BT, Ruiz-Hernandez E, Harkin A: Intranasal Polymeric and Lipid-Based Nanocarriers for CNS Drug Delivery. Pharmaceutics 2023; 15 Fonseca LC, Lopes JA, Vieira J, Viegas C, Oliveira CS, Hartmann RP et al: Intranasal drug delivery for treatment of Alzheimer's disease. Drug Deliv Transl Res 2021; 11: 411–425 Hanson LR, Frey WH, 2nd: Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci 2008; 9 Suppl 3: S5 Vera R, Hong N, Jiang B, Liang G, Eckenhoff MF, Kincaid HJ et al: Effects of Intranasal Dantrolene Nanoparticles on Brain Concentration and Behavior in PS19 Tau Transgenic Mice. J Alzheimers Dis 2024 Garbincius JF, Elrod JW: Mitochondrial calcium exchange in physiology and disease. Physiol Rev 2022; 102: 893–992 Gleitze S, Ramirez OA, Vega-Vasquez I, Yan J, Lobos P, Bading H et al: Ryanodine Receptor Mediated Calcium Release Contributes to Ferroptosis Induced in Primary Hippocampal Neurons by GPX4 Inhibition. Antioxidants (Basel) 2023; 12 Triantafilou K, Hughes TR, Triantafilou M, Morgan BP: The complement membrane attack complex triggers intracellular Ca2 + fluxes leading to NLRP3 inflammasome activation. J Cell Sci 2013; 126: 2903–13 Wongpun J, Chanmanee T, Tocharus J, Chokchaisiri R, Chaichompoo W, Suksamrarn A et al: The potential effects of festidinol treatment against the NLRP3 inflammasome and pyroptosis in D-galactose and aluminum chloride-induced Alzheimer's-like pathology in mouse brain. Int Immunopharmacol 2023; 119: 110181 Wan T, Li X, Fu M, Gao X, Li P, Guo W: NLRP3-Dependent Pyroptosis: A Candidate Therapeutic Target for Depression. Front Cell Neurosci 2022; 16: 863426 Fountoulakis KN, Saitis A, Schatzberg AF: Esketamine Treatment for Depression in Adults: A PRISMA Systematic Review and Meta-Analysis. Am J Psychiatry 2025: appiajp20240515 Jiang B, Shi Y, Abou MB, Xu L, Liang G, Wei H: Effects of chronic intranasal dantrolene on nasal mucosa morphology in mice. Eur Rev Med Pharmacol Sci 2022; 26: 198–203 Hasko G, Szabo C, Nemeth ZH, Lendvai B, Vizi ES: Modulation by dantrolene of endotoxin-induced interleukin-10, tumour necrosis factor-alpha and nitric oxide production in vivo and in vitro. Br J Pharmacol 1998; 124: 1099–106 Zhang IX, Herrmann A, Leon J, Jeyarajan S, Arunagiri A, Arvan P et al: ER stress increases expression of intracellular calcium channel RyR1 to modify Ca(2+) homeostasis in pancreatic beta cells. J Biol Chem 2023; 299: 105065 Abais JM, Xia M, Zhang Y, Boini KM, Li PL: Redox regulation of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid Redox Signal 2015; 22: 1111–29 Additional Declarations Yes Huafeng Wei is an inventor for patents application in USA and other countries, proposing intranasal dantrolene nanoparticles to treat dementia and depression in Alzheimer's disease. 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Louis","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lauren","middleName":"St.","lastName":"Louis","suffix":""},{"id":434705963,"identity":"62b5b579-ce09-49f2-a331-5f1c8491e8e3","order_by":6,"name":"Yutong Yi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yutong","middleName":"","lastName":"Yi","suffix":""},{"id":434705964,"identity":"76fbe429-449d-4656-8a86-f473638e2dc4","order_by":7,"name":"Ge Liang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2025-03-18 15:50:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6254774/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6254774/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41398-026-03816-x","type":"published","date":"2026-02-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79831377,"identity":"bd5212a5-7d15-4fc4-a7d2-fc2770be7197","added_by":"auto","created_at":"2025-04-03 10:34:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1005078,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/bf964aa921fb46b8cc1959be.jpg"},{"id":79832126,"identity":"327a5463-4741-4e18-8dc5-9a41540037e8","added_by":"auto","created_at":"2025-04-03 10:42:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":987216,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/49a4fa01e0e6339c0fbdd62c.jpg"},{"id":79831380,"identity":"079a5abe-a1f0-45f2-9397-b90d4ce08bbc","added_by":"auto","created_at":"2025-04-03 10:34:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":988812,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/933e5114a3731355e4cf4092.jpg"},{"id":79831379,"identity":"47018a12-1fec-4352-98cb-796ca71decd4","added_by":"auto","created_at":"2025-04-03 10:34:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":930678,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/8987697047938c3a3e479d1c.jpg"},{"id":79831381,"identity":"4878472c-c6c0-4b7b-a619-e2c93ce30f1d","added_by":"auto","created_at":"2025-04-03 10:34:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":901521,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/52e8e0ec8f4d7ffda421083c.jpg"},{"id":103119153,"identity":"6c54ac03-e1e3-49be-94ba-d27ae52eff7c","added_by":"auto","created_at":"2026-02-21 08:08:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5875155,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/60277826-6d44-4b18-a3a6-faf9db683644.pdf"},{"id":79831387,"identity":"01019284-a5f7-4321-b387-a337f9e445c6","added_by":"auto","created_at":"2025-04-03 10:34:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6738501,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"1Supplementaryinformationwtonly2162025Final1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6254774/v1/d322b2b79699ee7b0ffd3928.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e\nHuafeng Wei is an inventor for patents application in USA and other countries, proposing intranasal dantrolene nanoparticles to treat dementia and depression in Alzheimer's disease.","formattedTitle":"Intranasal dantrolene nanoparticles inhibit lipopolysaccharide-induced depression and anxiety behavior in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThree hundred million people worldwide suffer from major depressive disorder (MDD)\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. MDD is a chronic and recurrent disease affecting about 20% of the population and the leading cause of suicide\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. MDD imposes a tremendous psychological burden, as well as significant social repercussions and contributions to other disabilities\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. The expected direct and indirect costs of MDD are up to \u003cspan\u003e$\u003c/span\u003e6 trillion in the USA alone\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. An estimated 4% of the global population currently experience an anxiety disorder. In 2019, 301\u0026nbsp;million people in the world had an anxiety disorder, with a lifetime prevalence of approximately 34 percent\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. Major depression disorder and anxiety disorders were approximately twice as prevalent among women\u003csup\u003e \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e \u003c/sup\u003e, with overall age-specific rates remaining relatively stable or increasing across the lifespan\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. In 2021, an estimated 5.8\u0026nbsp;million (9.4%) children and adolescents were impacted by anxiety\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e. Depression and anxiety often co-exist and are the most common psychiatric diseases, which are chronic diseases with unclear mechanisms of pathology\u003csup\u003e \u003cb\u003e \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e \u003c/b\u003e \u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eClassical treatment of depression or anxiety psychiatric disorders includes atypical antipsychotics that modulate dopamine and serotonin neurotransmission\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Pharmacotherapies for anxiety disorders also include drugs that interact with GABA neurotransmission systems\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Unfortunately, antidepressants have significant limitations, including slow onset of action, high rates of nonresponse, and acute worsening of anxiety\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Benzodiazepines are not recommended for long-term use in some anxiety disorders, due to concerns about their potential for abuse, tolerance, and withdrawal, and they are ineffective in some anxiety spectrum disorders\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Treatment of MDD is prone to a high risk of resistance (up to 30% of patients are unresponsive to the first treatment) and relapse (up to 8%)\u003csup\u003e\u003cb\u003e3,10\u003c/b\u003e\u003c/sup\u003e. Treatment-resistant anxiety with remission rates may be as low as 25\u0026ndash;35%, and relapse rates post remission may be 30% after 10 years\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Thus, there is an urgent need to develop novel approaches to treat depression and anxiety, especially treatment-resistant depression, or anxiety, with minimal side effects or organ toxicity.\u003c/p\u003e \u003cp\u003eIncreasing studies indicated that glutamate neurotransmission plays a critical role in mood function and its imbalance may cause psychiatric disorders\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Glutamate is linked to the development of anxiety disorders through its regulation of neuropeptides, fear extinction, and stress response. Glutamate is also important in synaptic and neural plasticity related to psychiatric disorders. Ketamine, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, was approved in 1970 by the Food and Drug Administration (FDA) for use in children and adults as an anesthetic. Recent studies indicate that ketamine is effective in treating MDD, especially treatment-resistant depression (TRD)\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Ketamine is also effective in treating anxiety disorders, even those resistant to traditional treatments\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. While traditional antidepressants can take weeks to months to have an effect, ketamine has rapid effects on mood and suicidality, with mood changes reported as early as the first 4 hours after treatment\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. As an antagonist of NMDAR, a primary glutamate receptor on the plasma membrane that causes Ca\u003csup\u003e2+\u003c/sup\u003e influx into the cytosol from extracellular space, ketamine has become a safe and broadly effective drug to treat depression or anxiety disorders, so is the intranasal esketamine\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This indicates that glutamate-mediated excitotoxicity and associated disruption of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis play a key role in the pathology of depression and anxiety disorders.\u003c/p\u003e \u003cp\u003eAlthough molecular mechanisms are unclear, increasing evidence suggests that upstream disruption of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis and associated downstream inflammation and synapse dysfunction play critical roles in MDD pathologies\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, as well as anxiety disorder\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The overactivation of ryanodine receptors (RyRs) in MDD and associated excessive Ca\u003csup\u003e2+\u003c/sup\u003e release from endoplasmic reticulum (ER) results in depletion of ER Ca\u003csup\u003e2+\u003c/sup\u003e and pathological elevation of cytosol and mitochondrial Ca\u003csup\u003e2+\u003c/sup\u003e concentrations, detrimental to synapse function and cell survival\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. RyRs overactivation have also been shown to increase anxious behavior\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Upstream Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation results in mitochondria dysfunction\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, mitochondrial and cellular oxidative stress\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, activation of inflammasomes, and cell or neuron death by pyroptosis\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. This results in the release of inflammatory cytokines (IL-1β and IL-18) and pathological inflammation-mediated cell death by pyroptosis\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Pathological cytokines, especially pyroptosis-related IL-18 play important roles in psychiatric disorders\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Gut dysbiosis and associated inflammation also contribute to pathology in depression and anxiety behaviors\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Thus, a drug that inhibits upstream Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation and then downstream pathological inflammation and programmed cell death by pyroptosis and synapse destruction is expected to treat MDD and anxiety disorder effectively.\u003c/p\u003e \u003cp\u003eDantrolene, a RyRs antagonist, is a US Food and Drug Administration-approved drug for the treatment of malignant hyperthermia, muscle spasms, and neuroleptic syndrome, with tolerable side effects and occasional liver toxicity at high doses\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Dantrolene, with its ability to inhibit the common upstream critical Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation, is neuroprotective against many neurodegenerative diseases, including cerebral ischemia\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, Huntington\u0026rsquo;s disease\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, spinocerebellar ataxia\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, amyotrophic lateral sclerosis\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, and seizures\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Dantrolene robustly inhibited up to 78% of NMDA-induced elevation of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e concentration in cerebral cortical neurons \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, suggesting it may have great potential to treat major depression disorder with a similar molecular mechanism of ketamine\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, which needs to be studied urgently.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the therapeutic effectiveness and the potential mechanisms of intranasal dantrolene nanoparticles on depression and anxiety behavior associated with pathological inflammation in adult mice. We induced inflammation by intraperitoneal injection of LPS. Our results demonstrated that intranasal dantrolene nanoparticles for twelve consecutive weeks robustly inhibited LPS-induced depression and anxiety behaviors in adult mice. These beneficial effects of intranasal dantrolene nanoparticles treatment were associated with its robust and significant inhibition of LPS-induced pathological elevation of pyroptosis-related inflammation cytokine of IL-1β and IL-18 in blood and brains and loss of synapse proteins (PSD-95 and synpatin-1) in brains. This study provides proof of concept that intranasal dantrolene nanoparticles may be an effective treatment for depression and anxiety psychiatric disorders, potentially by inhibiting pathological inflammation and synapse destruction in the CNS.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania. All B6SJLF1/J adult mice were obtained from Jackson Lab. Mice were divided into different cages according to age and gender, with no more than five mice per cage. Both male and female mice were used in this study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental treatment groups\u003c/h3\u003e\n\u003cp\u003eAs demonstrated in supplemental Fig.\u0026nbsp;1, adult male and female mice (5\u0026ndash;10 months old) were randomly divided into different experimental groups. The mice were pretreated with either intranasal dantrolene nanoparticles (IN Dan, 5mg/kg) or vehicle (Veh), once a day, 5X/week (Monday through Friday). Control groups received no pretreatment. Fresh dantrolene nanoparticles were made every time before administration. Intranasal dantrolene nanoparticles stock solution was made at 5 mg/ml. The Ryanodex formulation vehicle was made fresh and contained all inactive ingredients in Ryanodex\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. At the end of the 4-week pretreatment period, mice were treated with a one-time intraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg). Mice in the control group, without pretreatment, were treated with LPS (LPS, 5mg/kg) once as well. The sham control group received no pretreatment or treatment. At 24 hours following the one-time LPS treatment, behavioral tests for depression or anxiety behaviors were performed on all mice, with the following order of least to most stressful tests: OPT, EPMT, TST, and FST. Thereafter, all mice were euthanized, and the blood or brain was harvested for examination of inflammation and programmed cell death by pyroptosis and synapse destruction in the blood and brains.\u003c/p\u003e \u003cp\u003eIntranasal dantrolene nanoparticle or vehicle administration\u003c/p\u003e \u003cp\u003eDantrolene (Sigma, St Louis, MO) was dissolved in the Ryanodex Formulation Vehicle (RFV: 125 mg mannitol, 25 mg polysorbate 80, 4mg povidone K12 in 5mL of sterile water and pH adjusted to 10.3), similar to our previous publications\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. For intranasal administration, the final concentration of dantrolene was 5 mg/mL as we have previously described. Mice were held and fixed by the scruff of their necks with one hand. With the other hand, mice were given a total of 1\u0026micro;L/gram of body weight of dantrolene nanoparticles or RFV. Accordingly, a mouse weighing 20 g would be given 20\u0026micro;L of solution, equal to a dantrolene dose at 5mg/kg. The solution was slowly delivered directly into the mouse\u0026rsquo;s nose. Care was taken to ensure that mice were minimally stressed, and that the solution remained in the nasal cavity and did not enter the stomach or lungs.\u003c/p\u003e\n\u003ch3\u003eForced swimming test (FST)\u003c/h3\u003e\n\u003cp\u003eDepression behavior was assessed using the FST, as described previously with some modification\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The mice from each group were settled in a clear glass tank of 25 cm height, 10 cm diameter, 15 cm water depth, and (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2) \u0026deg;C water temperature. The test's total period was 6 minutes, 2 minutes for adaptation, and the total immobility time was recorded in the next 4 minutes. Immobility occurred when the mice discontinued floundering on the surface of the water, instead appearing as floating in an equilibrium state. The greater the immobility time, the more severe the depression behavior.\u003c/p\u003e\n\u003ch3\u003eTail suspension test (TST)\u003c/h3\u003e\n\u003cp\u003eDepression behavior was assessed using the tail suspension test described previously, with modifications\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. We use specially manufactured tail suspension boxes, made of cartons with the dimensions (42 cm height X 18 cm width X 30 cm depth). To prevent animals from observing or interacting with each other, each mouse was suspended within its three-walled rectangular box. Each day, animals were acclimated to the testing room for at least 1 hour before the test. Each mouse was suspended in the middle of the box. The width and depth of the box were sufficiently sized to prevent the mouse from contacting the walls. In this setting, the approximate distance between the mouse's nose and the apparatus floor was 20\u0026ndash;25 cm. A plastic suspension bar (50 cm. height X 40 cm. depth), was used to suspend the tail of each mouse and positioned on the top of the box. At the bottom of each box, we placed a paper towel to collect feces or urine from the animals. A dark grey box was used for albino animals and a white colored box for mice of other coat colors. Before evaluation, the tail of the mouse was securely adhered to the suspension bar, which was able to withstand the mouse\u0026rsquo;s weight. A video camera was placed in position and started recording the TST session. The total duration of the test was 6 minutes. The paper towel was replaced after each trial. After all sessions were finished, Any-maze software (Stoelting, USA) was used to analyze the collected data.\u003c/p\u003e \u003cp\u003eDuring the behavioral analysis, the mobility time of each mouse was measured and subtracted from 360 seconds, producing the immobility time. The investigator was blinded to the animal groups. The higher immobility time, the more severe the depressive behavior.\u003c/p\u003e\n\u003ch3\u003eOpen field test (OFT)\u003c/h3\u003e\n\u003cp\u003eThe OFT was performed as previously described to evaluate anxiety behavior as described\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Each mouse was placed individually into the OFT apparatus (44 \u0026times; 44 \u0026times; 44 cm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e), facing the wall. The locomotor activity was recorded for six minutes with a camera above the apparatus under dim light. The anxiety behavior of each mouse was measured using the total distance traveled, center entries, immobility time, and time spent in the central zone. After each test, the apparatus was cleaned with 75% ethanol. Any-maze software (Stoelting USA) was used to analyze the collected data. The lesser the central zone distance and mean speed, the higher the anxiety behavior. The higher immobility time, the more severe the anxiety behavior.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eElevated plus maze test (EPMT)\u003c/h2\u003e \u003cp\u003eAnxiety was also assessed using EPMT, as described previously, although with some modification\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Mice were placed in the center of an elevated plus-maze (arms are 33 cm \u0026times; 5 cm, with 25 cm tall walls on the closed arms) under dim lighting and their behavior was videotaped for 5 minutes. We used Any-maze software (Stoelting USA) to analyze the collected data. The time spent in the closed and open arms, as well as the number of explorations of open arms, was measured and recorded. The more time spent in closed arms, the more severe the anxiety behavior.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEuthanasia and tissue collection\u003c/h3\u003e\n\u003cp\u003eMice were euthanized after the completion of all behavioral tests. As described previously\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, mice were deeply anesthetized with 2\u0026ndash;4% isoflurane delivered through a nose cone, with the concentration adjusted according to response to a toe pinch. The skin of each mouse was prepared, and an incision was made to open the chest and expose the heart. Blood was collected from the heart for the serum study using a syringe equipped with a 27G needle. The blood was centrifuged at 1400 rpm at 4◦C for 30 min, and the supernatant was collected and frozen at \u0026minus;\u0026thinsp;80◦C. The mice were euthanized by trans-cardial perfusion and exsanguination was conducted with cold phosphate-buffered saline.\u003c/p\u003e\n\u003ch3\u003eMeasurements of serum concentration of IL-1β and IL-18\u003c/h3\u003e\n\u003cp\u003eWe measured the serum IL-1β and IL-18 cytokines using an ELISA kit, following company instructions, and as we described previously for measurement of S100β, with some modification\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. All reagents and samples (the supernatant of the blood) were thawed to room temperature (18\u0026ndash;25\u0026deg;C) before use. It is recommended that all standards and samples be run at least in duplicate. Add 100 \u0026micro;L of each standard and sample into appropriate wells. Cover wells and incubate for 2.5 hours at room temperature or overnight at 4\u0026deg;C with gentle shaking. Discard the solution and wash it 4 times with 1X Wash Solution. Wash by filling each well with Wash Buffer (300 \u0026micro;L) using a multi-channel Pipette or auto washer. Complete removal of liquid at each step is essential to reliable performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. Add 100 \u0026micro;L of 1x prepared Detection Antibody to each well. Cover wells and incubate for 1 hour at room temperature with gentle shaking. Discard the solution. Repeat the wash procedure as in step 3. Add 100 \u0026micro;L of prepared Streptavidin solution to each well. Cover wells and incubate for 45 minutes at room temperature with gentle shaking. Discard the solution. Repeat the wash as in step 3. Add 100 \u0026micro;L of TMB One-Step Substrate Reagent (Item H) to each well. Cover wells and incubate for 30 minutes at room temperature in the dark with gentle shaking. Add 50 \u0026micro;L of Stop Solution (Item I) to each well. Read absorbance at 450 nm immediately. Mouse IL-1 beta ELISA Kit and Mouse IL18 ELISA Kit come from SIGMA(RAB0275-1KT/RAB08100-1KT).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting (Western Blot)\u003c/h2\u003e \u003cp\u003eAs we described previously\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, total brain tissues were extracted by homogenization using cold RIPA buffer (#9806S, Cell Technology, USA) supplemented with protease inhibitor cocktails (P8340 Roche). The brain homogenates were rocked at 4\u0026deg;C for 90 minutes and then centrifuged at 14 000 rpm (Brushless Microcentrifuge, Denville 260D) for 20 minutes at 4\u0026deg;C to remove cell debris. After collecting the supernatant, the protein concentration was measured using a BCA protein assay kit (Pierce, Rockford, IL 61101 USA). Briefly, equal amounts of protein (20\u0026micro;g/lane) were loaded onto 4\u0026ndash;20% gel electrophoresis of mini-protein TGX precast (Cat. #4561094, BIO-RAD) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, MERK Millipore, Ireland) using wet electrotransfer system (BIO-RAD, USA). Following the transfer membrane blocking with 5% BSA (Sigma-Aldrich) for 1 hour, then the PVDF membrane was incubated with primary antibodies overnight at 4\u0026deg;C including NLRP3, cleaved caspase-1, GSDMD-NT, IL-1β, IL-18, PSD95, Synapsin-1, GAPDH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Then, the membranes were incubated with a secondary antibody including anti-Mouse IgG1 HRP-linked; Anti-rabbit IgG, HRP-linked that was conjugated to horseradish peroxidase and washed with Tris-buffered saline containing 0.2% Tween-20 (TBST). Following that, TBST was used for washing the membranes three times for 10 minutes. After incubation with secondary antibodies, protein bands were visualized using ECL Western Blotting Detection Reagents (Cytiva, amersham, UK) and quantified for band intensity using ImageJ software (National Institutes of Health, Bethesda, MD, USA), by two persons blinded to treatments and averaged for each mouse.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCat. and Manufacture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDilution\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti- NLRP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68102-1-Ig; Proteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-Human procaspase-1/ cleaved caspase-1 p20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#2225S, Cell signaling Technology,USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti- Cleaved GSDMD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#36425S; Cell signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-IL-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31202S, Cell signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-IL-18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10663-1-AP; Proteintech\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:3 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-PSD95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#2507; Cell signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-Synapsin-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#5297; Cell signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMA5\u0026ndash;15738, Thermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:10 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary antibody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-Mouse IgG1; HRP -linked\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e# PA1-74421, Thermo Fisher Scientific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:10 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-rabbit IgG, HRP-linked\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e#7074; Cell signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:1 000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (Means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Statistical analyses were employed with GraphPad Prism (Version 9.3.1, CA, USA). Comparisons of more than two groups were conducted by one-way ANOVA with Tukey\u0026rsquo;s multiple comparison test. The N values in each group represent the number of mice. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIntranasal dantrolene nanoparticle pretreatment significantly inhibited LPS-induced helplessness and anxiety-related behaviors.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe FST and TST tests are commonly used to evaluate helplessness or depression behaviors in mice and to test the drug\u0026rsquo;s efficacy versus side effects\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In both FST (Fig.\u0026nbsp;1A) and TST (Fig.\u0026nbsp;1B), LPS induced significantly increased immobile times (helplessness or depression behaviors) by 89% (60.1 vs. 113.3) and 90% (98.1 vs. 186.6) respectively. Intranasal dantrolene nanoparticle pretreatment robustly inhibited the increased immobile time in FST by 41% (113.3 vs. 66.9) and in TST by 46% (186.6 vs. 101.5). In contrast, the intranasal dantrolene nanoparticles vehicle control did not demonstrate the same inhibitory effects on helplessness behaviors. The elevated plus maze test (EPMT) and open field test (OPT) are commonly used to evaluate anxiety-related behaviors in mice and test drug efficacy to treat anxiety behaviors\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. In both EPMT (Fig.\u0026nbsp;1C) and OFT (Fig.\u0026nbsp;1D), LPS induced significantly increased time stay in closed arm in EPMT (anxiety behaviors) by 46% (162 vs. 237) and immobile time in OPF by 130% (82 vs. 189) respectively. Intranasal dantrolene nanoparticle pretreatment robustly inhibited the increased time stay in closed arm in EPMT by 29% (237 vs. 169) and immobile time in OPT by 40% (189 vs. 114). In contrast, the intranasal dantrolene nanoparticles vehicle control did not demonstrate the same inhibitory effects on depression behaviors.\u003c/p\u003e \u003cp\u003eIn the open field test, the less central zone distance mice traveled and had a lower moving speed, the more severe the anxiety behavior (supplemental Fig.\u0026nbsp;2). Compared to the control without treatment, one-time LPS treatment significantly decreased mean speed (anxiety behavior) but not traveled zone distance. Intranasal dantrolene nanoparticles tended to inhibit LPS-induced decrease of travel zone distance and mean speed (Supplemental Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntranasal dantrolene nanoparticle pretreatment significantly inhibited LPS-induced helplessness and anxiety behaviors primarily in female mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further analyze the effects of sex on neuroprotective effects of intranasal dantrolene nanoparticles on helplessness and anxiety behavior. As demonstrated in Fig.\u0026nbsp;2, intranasal dantrolene nanoparticles but not its vehicle control primarily inhibited helpless behavior determined by both FST (Fig.\u0026nbsp;2A) and TST (Fig.\u0026nbsp;2B) in female but not male mice. For anxiety behavior, although intranasal dantrolene nanoparticle but not vehicle control inhibited LPS-induced anxiety behavior determined by EPMT (Fig.\u0026nbsp;2C) in male mice, the inhibitory effects of dantrolene on anxiety behaviors were most significant in female mice determined by both EPMT (Fig.\u0026nbsp;2C) and OFT (Fig.\u0026nbsp;2D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of pyroptosis-related inflammation cytokines in blood and brains.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared to control without treatment, IP injection of LPS (5mg/kg) for one time significantly increased blood concentrations of IL-1β by 147% (Fig.\u0026nbsp;3A, 136 vs. 336) and IL-18 by 67% (Fig.\u0026nbsp;3B, 233 vs. 390). Intranasal dantrolene nanoparticles but not its vehicle control significantly inhibited the increased IL-1β by 46% (336 vs. 183) and elevated IL-18 by 23% (Fig.\u0026nbsp;3B, 390 vs. 299). Furthermore, intranasal dantrolene nanoparticles significantly inhibited LPS-induced elevation of IL-1β and IL-18 proteins in both blood (Fig.\u0026nbsp;3A, B) and in brains (Fig.\u0026nbsp;3C, D, supplemental Figs.\u0026nbsp;3 and 4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntranasal dantrolene nanoparticles inhibited LPS-induced programmed cell death by pyroptosis in mice brains.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared to control mice without treatment, LPS treatment increased the protein levels of pyroptosis pathway activation (NLRP3, caspase-1 and N terminal GSDMD) in mice brains. Intranasal dantrolene nanoparticles significantly inhibited increase of Caspase-1 (P20), a biomarker of programmed cell death by pyroptosis (Fig.\u0026nbsp;4C, D, supplemental Fig.\u0026nbsp;6), and trended to inhibit LPS-induced elevation of pyroptosis pathway activation proteins (Fig.\u0026nbsp;4, A and B, supplemental Fig.\u0026nbsp;5, NLRP-3 and Fig.\u0026nbsp;4E and F, supplemental Fig.\u0026nbsp;7, N terminal GSDMD (GSDMD-NT))\u003c/p\u003e \u003cp\u003e \u003cb\u003eIntranasal dantrolene nanoparticles significantly inhibited LPS-induced synapse protein loss in the mice brains.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared to control without treatment, LPS significantly decreased the synapse proteins of PSD-95 by 45% (1.322 vs. 0.726, Fig.\u0026nbsp;5, A, C, supplemental Fig.\u0026nbsp;8) and synapsin-1 by 37% (0.925 vs. 0.579, Fig.\u0026nbsp;5, B, D, supplemental Fig.\u0026nbsp;9) in mice brains. Intranasal dantrolene nanoparticles but not vehicle control pretreatment for 4 weeks significantly inhibited the pathologically reduction of synapse proteins of PSD-95 by 77% (0.726 vs. 1.286, Fig.\u0026nbsp;5A, C, supplemental Fig.\u0026nbsp;8) and synpasin-1 by 76% (0.579 vs. 1.021, Fig.\u0026nbsp;5B and D, supplemental Fig.\u0026nbsp;9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is an urgent need for new therapeutic interventions to prevent or treat MDD\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, and anxiety\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e psychiatric disorders, especially those drug-resistant and recurrent depression and anxiety\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. This study has demonstrated that intranasal dantrolene nanoparticles are capable of ameliorating helplessness and anxiety behaviors in adult mice, associated with its ability to inhibit pathological elevation of pyroptosis related cytokines (IL-1β and IL-18) in blood, pyroptosis biomarker caspase-1 (P20) and synapse proteins loss in brains. Considering that upstream Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation plays an important role in the pathology of depression and anxiety, evidenced by the effectiveness of ketamine and esketamine in treating depression and anxiety resistant to traditional drug treatments\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, a new class of drugs, such as dantrolene, could be developed with a mechanism to correct the disruption of upstream Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation and associated downstream pathological inflammation and pyroptosis in psychiatric disorders.\u003c/p\u003e \u003cp\u003eIncreasing studies suggest that inflammation is an important pathology causing MDD and anxiety and could be a target for effective treatments\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR68 CR69\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Intracellular Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation has been proposed as the upstream cause of downstream mitochondria dysfunction, oxidative stress, and inflammation in MDD\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and anxiety\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Inhibition of upstream Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation through inhibiting over-activation of RyRs has been shown to suppress inflammation in Gulf War illness characterized by depression and dementia\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Although dantrolene has been shown to inhibit pathological inflammation with increased cytokines in diabetes\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, sepsis\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, and COVID-19 infection\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, its effects to inhibit Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation and associated inflammation for treating depression and anxiety behaviors have not yet been investigated until now and studied herewith.\u003c/p\u003e \u003cp\u003eSeveral animal models to establish depression and anxiety behaviors have been suggested based on the proposed neurobiology mechanisms, including altered neurotransmission, HPA axis abnormalities involved in chronic stress, inflammation, reduced neuroplasticity, and network dysfunction\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR81\" citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Induction of inflammation in animals has been increasingly used to establish depression and anxiety animal models, especially using LPS to induce inflammation, depression, and anxiety behaviors to test drug treatment efficacy\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan additionalcitationids=\"CR84 CR85\" citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The common approach of LPS administration is intraperitoneal injection because of its ease and feasibility. The dose and duration of IP injection of LPS to establish depression or anxiety behaviors has been varied but appears to be dose dependent. The high dose of LPS at 5 mg/kg, administered once, seems to be dependable in inducing inflammation and establishing depression behavior\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our study confirmed the efficiency of a one-time administration of IP LPS (5 mg/kg) to induce inflammation and depression and anxiety behavior in adult mice consistently. Accordingly, the LPS-induced inflammation-mediated depression behaviors are adequate to evaluate the therapeutic effects of dantrolene to treat depression. Our results demonstrated robust inhibition of LPS-induced inflammation and associated helplessness and anxiety behaviors by intranasal dantrolene nanoparticles pretreatment in adult mice. In addition to the protective effects of dantrolene against memory loss in various AD animal models\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan additionalcitationids=\"CR88\" citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, intranasal dantrolene nanoparticles also significantly inhibited helplessness and anxiety behaviors, and associated inflammation and pyroptosis, suggesting that pathological inflammation and pyroptosis play a critical role in both cognitive dysfunction and psychiatric disorders. Drugs that target inflammation pathology should be developed to treat both memory loss and psychiatric disorders in AD patients. Nevertheless, intranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of pyroptosis related inflammation cytokines (IL-1β and IL-18) in blood and ameliorated both depression and anxiety behavior in adult mice, suggesting that intranasal dantrolene nanoparticles could be developed as an effective drug to treat depression and/or anxiety in major depression disorder (MDD) patients potentially by its inhibition of inflammation and associated programmed cell death by pyroptosis.\u003c/p\u003e \u003cp\u003eMDD affects female patients twice as much as male\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e,\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. The proposed mechanisms for this sex difference include the effects of estrogen\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e, serotonin, and tryptophan metabolism\u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e, different stress reactions\u003csup\u003e\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e, etc. An important finding in this study is that intranasal dantrolene nanoparticles are more effective in ameliorating helplessness and anxiety behaviors in female than in male mice. Our result is consistent with MDD being more prevalent in female mice than in male. Also, the more significant inhibition of inflammation associated with helplessness and anxiety behavior in female mice by intranasal dantrolene nanoparticles suggested that dantrolene may inhibit critical pathology and etiology contributing to depression and anxiety behaviors. Although the exact mechanism is unclear, we propose that dantrolene inhibited the upper stream critical Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation and associated inflammation and associated programmed neuron death by pyroptosis and synapse protein loss.\u003c/p\u003e \u003cp\u003eIntranasal administration of drugs, especially in a nanoparticle formulation, significantly promotes drugs that bypass the blood-brain barrier (BBB) and penetrate the CNS, with reduced peripheral toxicity\u003csup\u003e\u003cb\u003e\u003cspan additionalcitationids=\"CR95\" citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our recent research work indicated that intranasal dantrolene nanoparticles achieved higher therapeutic efficacy in the brain compared to oral and subcutaneous forms of administration, with minimal or no side effects after chronic use\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Intranasal dantrolene nanoparticles significantly ameliorated memory loss in adult 5XFAD mice as a disease-modifying drug\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Increasing studies suggest ryanodine receptor overactivation and associated Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation is an upstream critical pathology leading to multiple downstream pathologies including mitochondria dysfunction\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, oxidative stresses\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, pathological inflammation\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, and neuron damage by pyroptosis\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Eventually, these pathologies result in depression and/or anxiety psychiatric disorders\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. This study demonstrated that inhibition of RyRs overactivation by dantrolene inhibited pathological inflammation, synapse proteins destruction, and ameliorated depression and anxiety behavior robustly. This study further strengthens the indication that upstream RyRs overactivation and Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation and associated downstream pathological inflammation and synapse destruction play important roles in depression and anxiety psychiatric disorders. Like esketamine\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e, the intranasal dantrolene nanoparticles could become an effective disease-modifying drug treatment for both depression and anxiety psychiatric disorders and need to be investigated in future clinical studies.\u003c/p\u003e \u003cp\u003eSince depression and anxiety psychiatric disorders are chronic diseases, they require long-term drug treatments. Accordingly, proposed drugs must limit side effects or organ toxicity with chronic treatment. The major advantage of using intranasal dantrolene nanoparticles, in comparison to commonly used oral or intravenous approaches, is that it significantly increases the brain/blood concentration ratio of dantrolene\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, especially in aged mice\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. This promotes its CNS therapeutic effects, while minimizing its side effects or organ toxicity\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. Our previous study demonstrated no side effects on nose structure and smell function\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, liver structure\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and function, or muscle function\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e after up to 10 months of chronic treatment in adult 5XFAD mice\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e, suggesting that intranasal dantrolene nanoparticles are safe to be used chronically in animals. Chronic use of dantrolene in human patients needs to be investigated further.\u003c/p\u003e \u003cp\u003eThis study has the following limitations: 1. The mice are from 5 to 10 months old. Although they are considered adult mice, future studies need to focus on different age groups, especially in aged mice, considering the high prevalence of psychiatric disorders in the aged population. 2). We were unable to measure the changes of cytosol versus mitochondria Ca\u003csup\u003e2+\u003c/sup\u003e levels in the brain tissue due to technological challenges. However, dantrolene has been shown to inhibit LPS or AD gene mutation-induced overactivation of RyRs and associated Ca\u003csup\u003e2+\u003c/sup\u003e dysregulation in different cell culture models\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e,\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. 3). We did not measure the contents of reactive oxygen species (ROS) concentrations in the brain, which is usually the upstream pathology to activate NLRP3 inflammasome\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. 4). We did not measure other biomarkers demonstrating effects of intranasal dantrolene nanoparticles on programmed cell death by pyroptosis in brains. 6). We did not study the dose-response of dantrolene to treat depression and anxiety behaviors. Physiological Ca\u003csup\u003e2+\u003c/sup\u003e release from ER is important for many physiological functions in cells, which means over-inhibition of calcium release from the ER may be harmful to cells, as we described previously. The adequate dose of intranasal dantrolene nanoparticles to treat psychiatric disorders must be investigated thoroughly before its clinical use in patients.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrated that intranasal dantrolene nanoparticles significantly ameliorated inflammation-mediated depression and anxiety behaviors in adult mice. This neuroprotective effect against depression and anxiety behaviors was associated with its robust inhibition of pathological elevation of pyroptosis-related inflammation cytokines in blood and the synapse destruction in the brains. This study may inspire future studies to repurpose intranasal dantrolene nanoparticles in treating depression and anxiety psychiatric disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interests\u003c/h2\u003e \u003cp\u003eDrs. Huafeng Wei and Ge Liang are listed as inventors of patent applications entitled \u0026ldquo;Intranasal Administration of Dantrolene for Treatment of Alzheimer\u0026rsquo;s Disease\u0026rdquo; in multiple countries by The Trustees of the University of Pennsylvania.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eH.W. conceived and designed the study. J.L., Y.L, P.B. J.G, L.S.L, Y.Y., G.L, H.W. conducted experiments, acquired and analyzed the data, J.L., Y.L, P.B. J.G, L.S.L, Y.Y., G.L, H.W. analyzed data and contributed to the manuscript preparation. H.W. wrote the manuscript. All the authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by grants to HW from the National Institute on Aging (R01AG061447) and NIA R01 Supplemental (3R01AG061447-03S1). The research was performed in the lab of Dr. Huafeng Wei and should be attributed to the Department of Anesthesiology, University of Pennsylvania. We appreciate the technical support from Rebecca Chae from Rowan University, New Jersey, U.S.A. and Kyulee Kim from UPENN, Pennsylvania, U.S.A.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHerrman H, Kieling C, McGorry P, Horton R, Sargent J, Patel V: Reducing the global burden of depression: a Lancet-World Psychiatric Association Commission. Lancet 2019; 393: e42-e43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRikhani K, Vas C, Jha MK: Approach to Diagnosis and Management of Treatment-Resistant Depression. Psychiatr Clin North Am 2023; 46: 247\u0026ndash;259\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelge JB, Sabbe ACF, Sabbe B: An update on pharmacotherapy for recurrent depression in 2022. Expert Opin Pharmacother 2023: 1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGururajan A, Reif A, Cryan JF, Slattery DA: The future of rodent models in depression research. Nat Rev Neurosci 2019; 20: 686\u0026ndash;701\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzuhany KL, Simon NM: Anxiety Disorders: A Review. JAMA 2022; 328: 2431\u0026ndash;2445\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarkas DA, Villeneuve AH, Daneshmend AZB, Villeneuve PJ, McQuaid RJ: Sex differences in the inflammation-depression link: A systematic review and meta-analysis. Brain Behav Immun 2024; 121: 257\u0026ndash;268\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAltemus M, Sarvaiya N, Neill Epperson C: Sex differences in anxiety and depression clinical perspectives. Front Neuroendocrinol 2014; 35: 320\u0026ndash;30\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSomers JM, Goldner EM, Waraich P, Hsu L: Prevalence and incidence studies of anxiety disorders: a systematic review of the literature. Can J Psychiatry 2006; 51: 100\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBruton AM, Wesemann DG, Machingo TA, Majak G, Johnstone JM, Marshall RD: Ketamine for mood disorders, anxiety, and suicidality in children and adolescents: a systematic review. Eur Child Adolesc Psychiatry 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAit Tayeb AEK, Poinsignon V, Chappell K, Bouligand J, Becquemont L, Verstuyft C: Major Depressive Disorder and Oxidative Stress: A Review of Peripheral and Genetic Biomarkers According to Clinical Characteristics and Disease Stages. Antioxidants (Basel) 2023; 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCowen PJ: SSRIs in the Treatment of Depression: A Pharmacological CUL-DE-SAC? Curr Top Behav Neurosci 2024; 66: 1\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalena K, Rao S, Mosler C: Review of Current Clinical Options for the Management of Behavioral and Psychological Symptoms of Dementia. Sr Care Pharm 2024; 39: 300\u0026ndash;310\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuss P, Ferreri F, Bourin M: An update on the anxiolytic and neuroprotective properties of etifoxine: from brain GABA modulation to a whole-body mode of action. Neuropsychiatr Dis Treat 2019; 15: 1781\u0026ndash;1795\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaganin W, Signorini S, Leccese V, Sciarretta A: [Treatment-resistant depression. From classification to new therapies.]. Riv Psichiatr 2022; 57: 258\u0026ndash;272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson S, Peacock EE, Scott R: Benzodiazepine Use Disorder: Common Questions and Answers. Am Fam Physician 2023; 108: 260\u0026ndash;266\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabaac BJ, Shinozuka K, Arenas A, Beutler BD, Cherian K, Evans VD et al: Psychedelic Therapy: A Primer for Primary Care Clinicians-Historical Perspective and Overview. Am J Ther 2024; 31: e97-e103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarawagi A, Soni ND, Patel AB: Glutamate and GABA Homeostasis and Neurometabolism in Major Depressive Disorder. Front Psychiatry 2021; 12: 637863\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen L: The mechanistic basis for the rapid antidepressant-like effects of ketamine: From neural circuits to molecular pathways. Prog Neuropsychopharmacol Biol Psychiatry 2024; 129: 110910\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosti S: Ketamine for Major Depressive Disorder. Curr Top Behav Neurosci 2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTully JL, Dahlen AD, Haggarty CJ, Schioth HB, Brooks S: Ketamine treatment for refractory anxiety: A systematic review. Br J Clin Pharmacol 2022; 88: 4412\u0026ndash;4426\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatveychuk D, Thomas RK, Swainson J, Khullar A, MacKay MA, Baker GB et al: Ketamine as an antidepressant: overview of its mechanisms of action and potential predictive biomarkers. Ther Adv Psychopharmacol 2020; 10: 2045125320916657\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosso G, d'Andrea G, Barlati S, Di Nicola M, Andriola I, Marcatili M et al: Esketamine Treatment Trajectory of Patients with Treatment-Resistant Depression in the Mid and Long-Term Run: Data from REAL-ESK Study Group. Curr Neuropharmacol 2025; 23: e1570159X337670\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeutschenbaur L, Beck J, Kiyhankhadiv A, Muhlhauser M, Borgwardt S, Walter M et al: Role of calcium, glutamate and NMDA in major depression and therapeutic application. Prog Neuropsychopharmacol Biol Psychiatry 2016; 64: 325\u0026ndash;33\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFries GR, Saldana VA, Finnstein J, Rein T: Molecular pathways of major depressive disorder converge on the synapse. Mol Psychiatry 2023; 28: 284\u0026ndash;297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarrosa-Jimenez J, Sanchez Carro Y, Ovejero-Benito MC, Del Sastre E, Garcia AG, Lopez MG et al: Intracellular calcium and inflammatory markers, mediated by purinergic stimulation, are differentially regulated in monocytes of patients with major depressive disorder. Neurosci Lett 2021; 765: 136275\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValiati FE, Feiten JG, Gea LP, Silveira EMJ, Scotton E, Caldieraro MA et al: Inflammation and damage-associated molecular patterns in major psychiatric disorders. Trends Psychiatry Psychother 2022\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wu LH, Hou F, Wang ZJ, Wu MN, Holscher C et al: Mitochondrial calcium uniporter knockdown in hippocampal neurons alleviates anxious and depressive behavior in the 3XTG Alzheimer's disease mouse model. Brain Res 2024; 1840: 149060\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhillips KF, Santos E, Blair RE, Deshpande LS: Targeting Intracellular Calcium Stores Alleviates Neurological Morbidities in a DFP-Based Rat Model of Gulf War Illness. Toxicol Sci 2019; 169: 567\u0026ndash;578\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCardon I, Grobecker S, Kucukoktay S, Bader S, Jahner T, Nothdurfter C et al: Mitochondrial and Cellular Function in Fibroblasts, Induced Neurons, and Astrocytes Derived from Case Study Patients: Insights into Major Depression as a Mitochondria-Associated Disease. Int J Mol Sci 2024; 25\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong Y, Cao H, Zuo C, Gu Z, Huang Y, Miao J et al: Mitochondrial dysfunction: A fatal blow in depression. Biomed Pharmacother 2023; 167: 115652\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindqvist D, Dhabhar FS, James SJ, Hough CM, Jain FA, Bersani FS et al: Oxidative stress, inflammation and treatment response in major depression. Psychoneuroendocrinology 2017; 76: 197\u0026ndash;205\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou XY, Thai M, Roediger D, Mueller BA, Cullen KR, Klimes-Dougan B et al: Mitochondrial health, NLRP3 inflammasome activation, and white matter integrity in adolescent mood disorders: A pilot study. J Affect Disord 2023; 340: 149\u0026ndash;159\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao H, Yang D, Nie K, Lin R, Peng L, Zhou X et al: Hesperidin may improve depressive symptoms by binding NLRP3 and influencing the pyroptosis pathway in a rat model. Eur J Pharmacol 2023; 952: 175670\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Sun Y, Song M, Song Y, Fang Y, Zhang Q et al: NLRP3/caspase-1/GSDMD-mediated pyroptosis exerts a crucial role in astrocyte pathological injury in mouse model of depression. JCI Insight 2021; 6\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimon MS, Schiweck C, Arteaga-Henriquez G, Poletti S, Haarman BCM, Dik WA et al: Monocyte mitochondrial dysfunction, inflammaging, and inflammatory pyroptosis in major depression. Prog Neuropsychopharmacol Biol Psychiatry 2021; 111: 110391\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaufmann FN, Costa AP, Ghisleni G, Diaz AP, Rodrigues ALS, Peluffo H et al: NLRP3 inflammasome-driven pathways in depression: Clinical and preclinical findings. Brain Behav Immun 2017; 64: 367\u0026ndash;383\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVogelzangs N, de Jonge P, Smit JH, Bahn S, Penninx BW: Cytokine production capacity in depression and anxiety. Transl Psychiatry 2016; 6: e825\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang LM, Zhang DX, Song RX, Lv JM, Wang LY, Wu ZY et al: IL-18BP Alleviates Anxiety-Like Behavior Induced by Traumatic Stress via Inhibition of the IL-18R-NLRP3 Signaling Pathway in a Mouse Model of Hemorrhagic Shock and Resuscitation. Mol Neurobiol 2023; 60: 382\u0026ndash;394\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatt S, Kanoujia J, Mohana Lakshmi S, Patil CR, Gupta G, Chellappan DK et al: Role of Brain-Gut-Microbiota Axis in Depression: Emerging Therapeutic Avenues. CNS Neurol Disord Drug Targets 2023; 22: 276\u0026ndash;288\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnand N, Gorantla VR, Chidambaram SB: The Role of Gut Dysbiosis in the Pathophysiology of Neuropsychiatric Disorders. Cells 2022; 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatto D, Joyner RW: Dantrolene, StatPearls. Treasure Island (FL), 2020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakayama R, Yano T, Ushijima K, Abe E, Terasaki H: Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CA1 region subjected to transient ischemia. Anesthesiology 2002; 96: 705\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei HF, Perry DC: Dantrolene is cytoprotective in two models of neuronal cell death. Journal of Neurochemistry 1996; 67: 2390\u0026ndash;2398\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Wu J, Lvovskaya S, Herndon E, Supnet C, Bezprozvanny I: Dantrolene is neuroprotective in Huntington's disease transgenic mouse model. Mol Neurodegener 2011; 6: 81\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Tang TS, Tu H, Nelson O, Pook M, Hammer R et al: Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3. J Neurosci 2008; 28: 12713\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRothstein JD, Kuncl RW: Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity. J Neurochem 1995; 65: 643\u0026ndash;51\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoyero PX, Higa GSV, Kostecki DS, Dos Santos BA, Almeida C, Andrade KA et al: Ryanodine receptors drive neuronal loss and regulate synaptic proteins during epileptogenesis. Exp Neurol 2020; 327: 113213\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeshavarz M, Fotouhi M, Rasti A: Dantrolene: A Selective Ryanodine Receptor Antagonist, Protects Against Pentylenetetrazole-Induced Seizure in Mice. Acta Med Iran 2016; 54: 555\u0026ndash;561\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrandsen A, Schousboe A: Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate and kainate in cultured cerebral cortical neurons. Proc Natl Acad Sci U S A. 1992; 89: 2590\u0026ndash;2594\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Cheng L, Ma J, Yuan J, Pi C, Xiong L et al: Molecular mechanisms of rapid-acting antidepressants: New perspectives for developing antidepressants. Pharmacol Res 2023; 194: 106837\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVera R, Hong N, Jiang B, Liang G, Eckenhoff MF, Kincaid HJ et al: Effects of intranasal dantrolene nanoparticles on brain concentration and behavior in PS19 tau transgenic mice. Res Sq 2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Y, Zhang L, Gao X, Zhang J, Ben Abou M, Liang G et al: Intranasal Dantrolene as a Disease-Modifying Drug in Alzheimer 5XFAD Mice. J Alzheimers Dis 2020; 76: 1375\u0026ndash;1389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Shi Y, Yu S, Wang Y, Meng Q, Liang G et al: Intranasal administration of dantrolene increased brain concentration and duration. PLoS One 2020; 15: e0229156\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUeno H, Takahashi Y, Murakami S, Wani K, Matsumoto Y, Okamoto M et al: Effect of simultaneous testing of two mice in the tail suspension test and forced swim test. Sci Rep 2022; 12: 9224\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCan A, Dao DT, Terrillion CE, Piantadosi SC, Bhat S, Gould TD: The tail suspension test. J Vis Exp 2012: e3769\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones SK, McCarthy DM, Vied C, Stanwood GD, Schatschneider C, Bhide PG: Transgenerational transmission of aspartame-induced anxiety and changes in glutamate-GABA signaling and gene expression in the amygdala. Proc Natl Acad Sci U S A 2022; 119: e2213120119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho WH, Noh K, Lee BH, Barcelon E, Jun SB, Park HY et al: Hippocampal astrocytes modulate anxiety-like behavior. Nat Commun 2022; 13: 6536\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoseph DJ, Liu C, Peng J, Liang G, Wei H: Isoflurane mediated neuropathological and cognitive impairments in the triple transgenic Alzheimer's mouse model are associated with hippocampal synaptic deficits in an age-dependent manner. PLoS One 2019; 14: e0223509\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang G, Ward C, Peng J, Zhao YF, Huang BS, Wei HF: Isoflurane Causes Greater Neurodegeneration Than an Equivalent Exposure of Sevoflurane in the Developing Brain of Neonatal Mice. Anesthesiology 2010; 112: 1325\u0026ndash;1334\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Liang G, Liang S, Mund R, Shi Y, Wei H: Dantrolene Ameliorates Impaired Neurogenesis and Synaptogenesis in Induced Pluripotent Stem Cell Lines Derived from Patients with Alzheimer's Disease. Anesthesiology 2020; 132: 1062\u0026ndash;1079\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu QF, Choi H, Son T, Kim YM, Kanmani S, Chin YW et al: Co-Treatment with the Herbal Medicine SIP3 and Donepezil Improves Memory and Depression in the Mouse Model of Alzheimer's Disease. Curr Alzheimer Res 2022; 19: 246\u0026ndash;263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmada N, Hirose T, Suzuki M, Kakumoto Y, Futamura T, Maeda K et al: Synergistic anti-depressive effect of combination treatment of Brexpiprazole and selective serotonin reuptake inhibitors on forced swimming test in mice. Neuropsychopharmacol Rep 2023; 43: 132\u0026ndash;136\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarkonyi D, Torok B, Sipos E, Fazekas CL, Banrevi K, Correia P et al: Investigation of Anxiety- and Depressive-like Symptoms in 4- and 8-Month-Old Male Triple Transgenic Mouse Models of Alzheimer's Disease. Int J Mol Sci 2022; 23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePentkowski NS, Rogge-Obando KK, Donaldson TN, Bouquin SJ, Clark BJ: Anxiety and Alzheimer's disease: Behavioral analysis and neural basis in rodent models of Alzheimer's-related neuropathology. Neurosci Biobehav Rev 2021; 127: 647\u0026ndash;658\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrady LS, Lisanby SH, Gordon JA: New directions in psychiatric drug development: promising therapeutics in the pipeline. Expert Opin Drug Discov 2023: 1\u0026ndash;16\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJha MK, Mathew SJ: Pharmacotherapies for Treatment-Resistant Depression: How Antipsychotics Fit in the Rapidly Evolving Therapeutic Landscape. Am J Psychiatry 2023; 180: 190\u0026ndash;199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Q, Li W, Chen P, Wang L, Bao X, Huang R et al: Microglial NLRP3 inflammasome-mediated neuroinflammation and therapeutic strategies in depression. Neural Regen Res 2024; 19: 1890\u0026ndash;1898\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu WJ, Hu T, Jiang CL: Cool the Inflamed Brain: A Novel Anti-inflammatory Strategy for the Treatment of Major Depressive Disorder. Curr Neuropharmacol 2024; 22: 810\u0026ndash;842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChin Fatt CR, Mayes TL, Trivedi MH: Immune Dysregulation in Treatment-Resistant Depression: Precision Approaches to Treatment Selection and Development of Novel Treatments. Psychiatr Clin North Am 2023; 46: 403\u0026ndash;413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo B, Zhang M, Hao W, Wang Y, Zhang T, Liu C: Neuroinflammation mechanisms of neuromodulation therapies for anxiety and depression. Transl Psychiatry 2023; 13: 5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArioz BI, Tastan B, Tarakcioglu E, Tufekci KU, Olcum M, Ersoy N et al: Melatonin Attenuates LPS-Induced Acute Depressive-Like Behaviors and Microglial NLRP3 Inflammasome Activation Through the SIRT1/Nrf2 Pathway. Front Immunol 2019; 10: 1511\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eResende R, Fernandes T, Pereira AC, De Pascale J, Marques AP, Oliveira P et al: Mitochondria, endoplasmic reticulum and innate immune dysfunction in mood disorders: Do Mitochondria-Associated Membranes (MAMs) play a role? Biochim Biophys Acta Mol Basis Dis 2020; 1866: 165752\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou GQ, Wang X, Gao P, Qin TZ, Guo L, Zhang ZW et al: Intestinal microbiota via NLRP3 inflammasome dependent neuronal pyroptosis mediates anxiety-like behaviour in mice exposed to 3.5 GHz radiofrequency radiation. Sci Total Environ 2024; 927: 172391\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark AL, Kanekura K, Lavagnino Z, Spears LD, Abreu D, Mahadevan J et al: Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death. Sci Rep 2017; 7: 5611\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson JD, Han Z, Otani K, Ye H, Zhang Y, Wu H et al: RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets. J Biol Chem 2004; 279: 24794\u0026ndash;802\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFischer DR, Sun X, Williams AB, Gang G, Pritts TA, James JH et al: Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats. Shock 2001; 15: 200\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHotchkiss RS, Karl IE: Dantrolene ameliorates the metabolic hallmarks of sepsis in rats and improves survival in a mouse model of endotoxemia. Proc Natl Acad Sci U S A. 1994; 91: 3039\u0026ndash;3043\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Kuraishy HM, Al-Gareeb AI, El-Bouseary MM, Alexiou A, Papadakis M, Batiha GE: Calpain/calpstatin pathway in COVID-19: Role of dantrolene. J Cell Mol Med 2024; 28: e18005\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlkazmi L, Al-Kuraishy HM, Al-Gareeb AI, El-Bouseary MM, Ahmed EA, Batiha GE: Dantrolene and ryanodine receptors in COVID-19:The daunting taskand neglected warden. Clin Exp Pharmacol Physiol 2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillas Boas GR, Boerngen de Lacerda R, Paes MM, Gubert P, Almeida W, Rescia VC et al: Molecular aspects of depression: A review from neurobiology to treatment. Eur J Pharmacol 2019; 851: 99\u0026ndash;121\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDean J, Keshavan M: The neurobiology of depression: An integrated view. Asian J Psychiatr 2017; 27: 101\u0026ndash;111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSapolsky RM: Psychiatric distress in animals versus animal models of psychiatric distress. Nat Neurosci 2016; 19: 1387\u0026ndash;1389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahdirejei HA, Peeri M, Azarbayjani MA, Fattahi Masrour F: Fluoxetine combined with swimming exercise synergistically reduces lipopolysaccharide-induced depressive-like behavior by normalizing the HPA axis and brain inflammation in mice. Pharmacol Biochem Behav 2023; 232: 173640\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuo R, Cheng X, Luo L, Yang L, Zhao Y, Zhou Y et al: Cinnamic Acid Improved Lipopolysaccharide-Induced Depressive-Like Behaviors by Inhibiting Neuroinflammation and Oxidative Stress in Mice. Pharmacology 2022; 107: 281\u0026ndash;289\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Ali T, He K, Liu Z, Shah FA, Ren Q et al: Ibrutinib alleviates LPS-induced neuroinflammation and synaptic defects in a mouse model of depression. Brain Behav Immun 2021; 92: 10\u0026ndash;24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Xu Y, Liu Z, Shi M, Zhang Y, Deng Y et al: TRPV4 inhibitor HC067047 produces antidepressant-like effect in LPS-induced depression mouse model. Neuropharmacology 2021; 201: 108834\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng J, Liang G, Inan S, Wu Z, Joseph DJ, Meng Q et al: Dantrolene ameliorates cognitive decline and neuropathology in Alzheimer triple transgenic mice. Neurosci Lett 2012; 516: 274\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakroborty S, Briggs C, Miller MB, Goussakov I, Schneider C, Kim J et al: Stabilizing ER Ca2\u0026thinsp;+\u0026thinsp;channel function as an early preventative strategy for Alzheimer's disease. PLoS One 2012; 7: e52056\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOules B, Del Prete D, Greco B, Zhang X, Lauritzen I, Sevalle J et al: Ryanodine receptor blockade reduces amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer disease. J Neurosci 2012; 32: 11820\u0026ndash;34\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShors TJ, Millon EM, Chang HY, Olson RL, Alderman BL: Do sex differences in rumination explain sex differences in depression? J Neurosci Res 2017; 95: 711\u0026ndash;718\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandez-Guasti A, Fiedler JL, Herrera L, Handa RJ: Sex, stress, and mood disorders: at the intersection of adrenal and gonadal hormones. Horm Metab Res 2012; 44: 607\u0026ndash;18\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIqbal J, Huang GD, Xue YX, Yang M, Jia XJ: Role of estrogen in sex differences in memory, emotion and neuropsychiatric disorders. Mol Biol Rep 2024; 51: 415\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSongtachalert T, Roomruangwong C, Carvalho AF, Bourin M, Maes M: Anxiety Disorders: Sex Differences in Serotonin and Tryptophan Metabolism. Curr Top Med Chem 2018; 18: 1704\u0026ndash;1715\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaher R, Moreno-Borrallo A, Jindal D, Mai BT, Ruiz-Hernandez E, Harkin A: Intranasal Polymeric and Lipid-Based Nanocarriers for CNS Drug Delivery. Pharmaceutics 2023; 15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca LC, Lopes JA, Vieira J, Viegas C, Oliveira CS, Hartmann RP et al: Intranasal drug delivery for treatment of Alzheimer's disease. Drug Deliv Transl Res 2021; 11: 411\u0026ndash;425\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanson LR, Frey WH, 2nd: Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci 2008; 9 Suppl 3: S5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVera R, Hong N, Jiang B, Liang G, Eckenhoff MF, Kincaid HJ et al: Effects of Intranasal Dantrolene Nanoparticles on Brain Concentration and Behavior in PS19 Tau Transgenic Mice. J Alzheimers Dis 2024\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarbincius JF, Elrod JW: Mitochondrial calcium exchange in physiology and disease. Physiol Rev 2022; 102: 893\u0026ndash;992\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGleitze S, Ramirez OA, Vega-Vasquez I, Yan J, Lobos P, Bading H et al: Ryanodine Receptor Mediated Calcium Release Contributes to Ferroptosis Induced in Primary Hippocampal Neurons by GPX4 Inhibition. Antioxidants (Basel) 2023; 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTriantafilou K, Hughes TR, Triantafilou M, Morgan BP: The complement membrane attack complex triggers intracellular Ca2\u0026thinsp;+\u0026thinsp;fluxes leading to NLRP3 inflammasome activation. J Cell Sci 2013; 126: 2903\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWongpun J, Chanmanee T, Tocharus J, Chokchaisiri R, Chaichompoo W, Suksamrarn A et al: The potential effects of festidinol treatment against the NLRP3 inflammasome and pyroptosis in D-galactose and aluminum chloride-induced Alzheimer's-like pathology in mouse brain. Int Immunopharmacol 2023; 119: 110181\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan T, Li X, Fu M, Gao X, Li P, Guo W: NLRP3-Dependent Pyroptosis: A Candidate Therapeutic Target for Depression. Front Cell Neurosci 2022; 16: 863426\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFountoulakis KN, Saitis A, Schatzberg AF: Esketamine Treatment for Depression in Adults: A PRISMA Systematic Review and Meta-Analysis. Am J Psychiatry 2025: appiajp20240515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang B, Shi Y, Abou MB, Xu L, Liang G, Wei H: Effects of chronic intranasal dantrolene on nasal mucosa morphology in mice. Eur Rev Med Pharmacol Sci 2022; 26: 198\u0026ndash;203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHasko G, Szabo C, Nemeth ZH, Lendvai B, Vizi ES: Modulation by dantrolene of endotoxin-induced interleukin-10, tumour necrosis factor-alpha and nitric oxide production in vivo and in vitro. Br J Pharmacol 1998; 124: 1099\u0026ndash;106\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang IX, Herrmann A, Leon J, Jeyarajan S, Arunagiri A, Arvan P et al: ER stress increases expression of intracellular calcium channel RyR1 to modify Ca(2+) homeostasis in pancreatic beta cells. J Biol Chem 2023; 299: 105065\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbais JM, Xia M, Zhang Y, Boini KM, Li PL: Redox regulation of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid Redox Signal 2015; 22: 1111\u0026ndash;29\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6254774/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6254774/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the therapeutic effectiveness of intranasal dantrolene nanoparticles pretreatment to inhibit lipopolysaccharide (LPS)-induced pathological inflammation and synapse destruction and depressive and anxiety behavior in mice. B6SJLF1/J adult mice were pretreated with intranasal dantrolene nanoparticles (dantrolene: 5mg/kg), daily, Monday to Friday, 5 days per week, for 4 weeks. Then, mice were treated with an intraperitoneal injection of LPS (5mg/kg) for one time. Behavioral tests for depression and anxiety were performed 24 hours after a one-time LPS injection. Biomarkers for pyroptosis-related inflammation cytokines (IL-1β and IL-18) in the blood and brain were measured using enzyme-linked immunosorbent assay (ELISA) and immunoblotting, respectively. The changes of primary proteins activation inflammatory pyroptosis (NLRP3: NLR family pyrin domain containing 3, Caspase-1, N-GSDMD: N terminal protein gasdermin D) and synapse proteins (PSD-95 and synpatin-1) in brains were measured using immunoblotting. Intranasal dantrolene nanoparticles robustly inhibited LPS-induced depression and anxiety behavior. Intranasal dantrolene nanoparticles significantly inhibited LPS-induced pathological elevation of IL-1β and IL-18 in the blood and brain and inhibited LPS-induced activation of pyroptosis. Intranasal dantrolene nanoparticles significantly ameliorated decrease of PSD-95 and synpatin-1 proteins in brains. Thus, intranasal dantrolene nanoparticles have demonstrated neuroprotection against inflammation-mediated depression and anxiety behaviors and should be studied further as a future effective drug treatment of major depression disorder or anxiety psychiatric disorder.\u003c/p\u003e","manuscriptTitle":"Intranasal dantrolene nanoparticles inhibit lipopolysaccharide-induced depression and anxiety behavior in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 10:34:23","doi":"10.21203/rs.3.rs-6254774/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-01T10:23:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-05T12:27:04+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-22T22:47:05+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-21T12:38:28+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-19T07:20:54+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-04-21T19:38:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-01T16:00:15+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-03-27T08:45:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T11:02:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T11:01:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2025-03-19T15:16:29+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-03-19T12:22:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e9fa6226-02ab-41bc-9c4a-15a75f4a4f67","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46286094,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"},{"id":46286095,"name":"Health sciences/Diseases/Psychiatric disorders"}],"tags":[],"updatedAt":"2026-02-21T08:07:57+00:00","versionOfRecord":{"articleIdentity":"rs-6254774","link":"https://doi.org/10.1038/s41398-026-03816-x","journal":{"identity":"translational-psychiatry","isVorOnly":false,"title":"Translational Psychiatry"},"publishedOn":"2026-02-03 05:00:00","publishedOnDateReadable":"February 3rd, 2026"},"versionCreatedAt":"2025-04-03 10:34:23","video":"","vorDoi":"10.1038/s41398-026-03816-x","vorDoiUrl":"https://doi.org/10.1038/s41398-026-03816-x","workflowStages":[]},"version":"v1","identity":"rs-6254774","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6254774","identity":"rs-6254774","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}