The Antioxidant Activity of Ketamine: Threshold-Dependent Mechanism in Treatment-Resistant Depression?

The Antioxidant Activity of Ketamine: Threshold-Dependent Mechanism in Treatment-Resistant Depression? | 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 Short Report The Antioxidant Activity of Ketamine: Threshold-Dependent Mechanism in Treatment-Resistant Depression? Zofia Winczewska, Magdalena Górska-Ponikowska, Wiesław Jerzy Cubała This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8624006/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aim of study: The antidepressant effect of ketamine may be at least partly due to its antioxidant activity. The aim of the study was to assess whether ketamine develops a protective effect on HT22 neuronal cells under conditions of oxidative stress (OS) induced by hydrogen peroxide (H 2 O 2 ). Material and methods The experiment was performed on an immortalized mouse hippocampal HT22 cell line. Cell viability after treatment with 25 ng/ml ketamine and 10 µM – 1000 µM H 2 O 2 for 24h was assessed by MTT assay. Results Ketamine at a concentration of 25 ng/mL increased the viability of HT22 neuronal cells under conditions of OS mimicked by H 2 O 2 , but only for a concentration of 1000 µM H 2 O 2 , which reflects the high level of OS typical of treatment-resistant depression (TRD). At 1000 µM H 2 O 2 , cell viability was 12% (± 1.63%), while after incubation with 1000 µM H 2 O 2 + 25 ng/ml ketamine, cell viability was 38% (± 9.12%). The results suggest that ketamine has a protective effect on HT22 cells, but that this effect is nonlinear and dependent on OS intensity, activated only at critical H 2 O 2 overload. Conclusions Preliminary results suggest that ketamine reverses the cytotoxicity of H 2 O 2 on HT22 cells, but only at specific, high concentrations of H 2 O 2 typical of severe subtypes of depression. This indicates a threshold, antioxidant mechanism of ketamine's antidepressant action, which could be used in further research on predictive models of response to ketamine treatment, which in the future could translate into individualized treatment and increased therapeutic success. treatment resistant depression ketamine oxidative stress hydrogen peroxide HT22 Figures Figure 1 INTRODUCTION TRD is a severe subtype of major depressive disorder (MDD), occurring in approximately 30% of patients who do not achieve clinical improvement despite multiple pharmacological interventions [ 1 ]. The lack of efficacy of antidepressants in a significant percentage of patients indicates that the mechanisms associated with symptom persistence in TRD go beyond the monoamine theory [ 2 ]. The role of OS in the etiopathogenesis of depression is particularly interesting and well established. One of the main mediators of OS is H 2 O 2 - in people with severe, recurrent depression, elevated H 2 O 2 concentrations are observed along with depletion of antioxidant defenses, including low levels of superoxide dismutase (SOD) [ 3 ]. Elevated H 2 O 2 concentrations are characteristic of individuals with recurrent, severe depression. A decrease in total antioxidant capacity (TAC) is observed, accompanied by an increase in total oxidation status (TOS) and markers of inflammation, including 8-OHdG [ 3 , 4 ]. The results of a recently published study by our team also showed high levels of H 2 O 2 in individuals with TRD, the source of which was addressed in abnormal estrogen metabolism inducing OS [ 5 ]. In view of the above data OS is positioned as an important factor in the development of TRD and responsible for the heterogeneity of the disease, which may be behind the different responses of patients to treatment [ 3 ]. Ketamine, classified as a rapid-acting antidepressant (RAAD) used in the treatment of TRD, produces a response in 50–70% of patients [ 6 ]. Although the mechanism of action of ketamine involves non-competitive antagonism of the N-methyl-D-aspartate (NMDAR) receptor, its antidepressant effect may be at least partly due to its antioxidant activity [ 7 ]. Preclinical studies have shown that ketamine administered under conditions of stress induced by repeated ACTH administration developed an antidepressant effect through an antioxidant mechanism - a decrease in TOS and malondialdehyde (MDA) was observed, with a simultaneous increase in superoxide dismutase (SOD) and paraoxonase 1 (PON-1) [ 7 ]. Ketamine also reduced oxidative damage in the HT22 cell model treated with glutamate, which induced OS in depression. An improvement in antioxidant status and neuroprotective effects via the cannabinoid receptor (CBD-1) were noted [ 8 ]. Interestingly, in models of traumatic brain injury, ketamine also showed a protective effect by reducing OS, including a decrease in MDA, an increase in SOD and glutathione peroxidase (GPx), and increased expression of antioxidant factors associated with the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway [ 9 ]. However, there are studies with contradictory results, which show that under stress-free conditions, ketamine led to an increase in MDA levels and thus a pro-inflammatory effect [ 7 ]. Another study also refers to the dual nature of ketamine, whose neuroprotective effect may be conditional and dependent on circumstances, including the dose of ketamine used [ 10 ]. The scientific literature confirms a varied response to ketamine in patients, which highlights the urgent need to individualize indications by identifying predictors of effective response to treatment [ 11 ]. MATERIAL AND METHODS Cell Culture Experiments employed immortalized mouse hippocampal cell line HT22 (kindly gifted by Tilman Grune from Institute of Nutrition, Department of Nutritional Toxicology, Friedrich Schiller University (Jena, Germany) and by Kelvin Davies from University of Southern California (USC, Los Angeles, CA, USA)). The cells were grown in Dulbecco’s Modified Eagle’s low glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich) at 37°C with 5% CO 2 . Cell Treatment HT22 cells were seeded in culture media at appropriate density 24 h before treatment. Next, the cells were treated with 25 ng/ml ketamine and H 2 O 2 in a concentration range of 10 µM – 1000 µM. The ketamine and H 2 O 2 solutions were prepared in sterile water. Cell Viability/Cell Proliferation Assay (MTT Assay) 10,000 HT22 cells per well were seeded in 96-well plates. After 24 hours, the cells were treated with 25 ng/ml ketamine and H 2 O 2 in a concentration range of 10 µM – 1000 µM for the next 24h. Solvent-treated control cells were regarded as 100% viable. MTT solution (Sigma-Aldrich, Poland) at a concentration of 0.5 mg/mL was used following incubation. The plates were centrifuged (700 g for 10 min) to extract the supernatant after 4 hours at 37°C. 100 µl of DMSO (Sigma-Aldrich, Poland) were used to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader (BioTek Instruments, Inc., USA). Percentage of the control was used to display the data. Each sample was prepared in three repetitions. Statistical analysis of the results The mean and standard deviation (SD) derived from three samples are used to present the results. The Student's t-test was used to evaluate the differences between the control samples and the ketamine-treated samples and to compare H 2 O 2 -treated samples to the one treated with the mixture of H 2 O 2 and ketamine. Statistical significance was established at a p-value of 0.05. Data analysis was done using GraphPad Prism (GraphPad Software, Inc., version 8, USA). RESULTS Effect of ketamine and H 2 O 2 on cell viability In order to evaluate the impact of ketamine and H 2 O 2 on HT22 cells, a cytotoxicity MTT assay was carried out. Precisely, HTT cell viability was determined after incubating the cells for 24 hours with 25 ng/ml ketamine and H 2 O 2 in a concentration range of 10 µM – 1000 µM. The sample's percentage of viable cells was determined in relation to the 100% viable control cells. HT22 cell viability was as follows: 34% (± 4.78%) after treatment with 25 ng/ml ketamine, 86% (± 3.66%) 10 µM H 2 O 2 , whereas 31% (± 0.85%) after incubation with the mixture 10 µM H 2 O 2 + 25 ng/ml ketamine. 30 µM H 2 O 2 decreased HT22 cell viability to 68% (± 3.26%), 30 µM H 2 O 2 + 25 ng/ml ketamine 34% (± 6.87%), 50 µM H 2 O 2 63% (± 3.45%), 50 µM H 2 O 2 + 25 ng/ml ketamine 35% (± 3.66%), 125 µM H 2 O 2 65% (± 10.78%), 125 µM H 2 O 2 + 25 ng/ml ketamine 36% (± 5.04%), 250 µM H 2 O 2 87% (± 6.03%), 250 µM H 2 O 2 + 25 ng/ml ketamine 40% (± 8.48%), 500 µM H 2 O 2 73% (± 4.62%), 500 µM H 2 O 2 + 25 ng/ml ketamine 39% (± 5.23%), 750 µM H 2 O 2 44% (± 1.46%), 750 µM H 2 O 2 + 25 ng/ml ketamine 31% (± 3.66%). In case of 1000 µM H 2 O 2 there were 12% (± 1.63%) viable, however after incubation with 1000 µM H 2 O 2 + 25 ng/ml ketamine cell viability was 38% (± 9.12%). (Fig. 1 ). DISCUSSION The results presented in this study are consistent with the scientific literature, which indicates that the antioxidant activity of ketamine may mediate its antidepressant effects. However in this study, we expanded the context of ketamine's antioxidant activity to include its potential threshold effect depending on the severity of depression, as the cytoprotective effect only became apparent at high concentrations of H 2 O 2 . Previous studies have consistently pointed to the antidepressant effect of ketamine through the modulation of signaling pathways associated with OS, including glutathione (GSH), Nrf2, SOD, and PON-1 [ 7 – 9 ]. It has also been suggested that the effectiveness of ketamine in people with TRD may be due to its beneficial effect on mitochondrial energy metabolism, whose dysfunction is associated with the development and progression of depression [ 12 ]. In a study by Weckmann et al., it was noted that after treatment with ketamine, the activation of the mTOR pathway increased, which led to an increase in the expression of genes related to energy metabolism and the promotion of mitochondrial biogenesis and redox balance regulation [ 12 ]. The high efficacy of ketamine treatment in patients with TRD may therefore result from the drug's beneficial effect on mitochondrial energy metabolism, which is closely linked to the glutamatergic system, whose increased activity plays a key role in the progression of depressive disorders [13]. In another study, the antidepressant effect of ketamine was linked to the induction of autophagy, inhibition of ferroptosis, and improvement of neuroplasticity, i.e., pathways closely related to OS and the persistence of depressive symptoms [ 14 ]. Furthermore, it has been suggested that ketamine may act differently in patients depending on specific subtypes of depression. One study indicated that ketamine was not effective in people with mild depression, suggesting that the effect of ketamine is modulated by the severity of the disease [15]. This study discusses a new, previously undescribed protective effect of ketamine on HT22 neuronal cells in which OS was induced by H 2 O 2 . Importantly, a threshold, conditional protective effect of ketamine was observed not at every level of H 2 O 2 exposure, but at specific concentrations corresponding to critical OS overload. These observations therefore suggest the existence of a certain ceiling in high ranges of H 2 O 2 concentrations at which the antioxidant mechanisms of ketamine are activated. Consistent with these results are observations of the opposite, pro-oxidative effect of ketamine under low stress conditions, suggesting variability in response to ketamine treatment potentially dependent on the biological context. These results deepen our understanding of the antioxidant mechanisms of ketamine and correspond with clinical observations suggesting that the effect of ketamine depends on the phenotype of depression. CONCLUSIONS AND FUTURE DIRECTIONS Numerous studies indicate that OS may influence the heterogeneity of depression and thus the varying response to treatment. Therefore, more and more studies are focusing on the search for biomarkers that would allow for targeted and effective treatment of TRD. The results presented in this study indicated that the cytoprotective effect of ketamine was only evident at high concentrations of H₂O₂, suggesting the existence of a potential threshold for the activation of antioxidant mechanisms and possibly explaining the varying response to treatment depending on biological background. These observations provide a promising basis for further research into the mechanisms of action of ketamine, as well as for clinical trials to assess whether the efficacy of ketamine correlates with the severity of OS and whether patients with higher H₂O₂ levels are indeed more likely to respond to treatment. This highlights the potential of H₂O₂ as a predictor of response to treatment in TRD, which may contribute to the personalization of therapy. Declarations CONFLICT OF INTEREST STATEMENT Wiesław Jerzy Cubała has received grants: Acadia, Alkermes, Allergan, Angelini, Auspex Pharmaceuticals, Beckley Psytech, BMS, Celon, Cephalon, Compass Pathways, Cortexyme, Ferrier, Forest Laboratories, GedeonRichter, GH Research, GWPharmaceuticals, HMNC Brain Health, IntraCellular Therapies, Janssen, KCR, Lilly, Lundbeck, MindMed, Minerva, MSD, NIH, Neumora, Novartis, Orion, Otsuka, Recognify Life Sciences, Sanofi, Seaport, Servier. Competing Interests Zofia Winczewska, Magdalena Górska-Ponikowska have no conflict of interests. Wiesław Jerzy Cubała has received grants: Acadia, Alkermes, Allergan, Angelini, Auspex Pharmaceuticals, Beckley Psytech, BMS, Celon, Cephalon, Compass Pathways, Cortexyme, Ferrier, Forest Laboratories, GedeonRichter, GH Research, GWPharmaceuticals, HMNC Brain Health, IntraCellular Therapies, Janssen, KCR, Lilly, Lundbeck, MindMed, Minerva, MSD, NIH, Neumora, Novartis, Orion, Otsuka, Recognify Life Sciences, Sanofi, Seaport, Servier. He has received honoraria: Adamed, Angelini, AstraZeneca, BMS, Celon, GH Research, GSK, Janssen, KRKA, Lekam, Lundbeck, Minerva, NeuroCog, Novartis, Orion, Pfizer, Polfa Tarchomin, Sanofi, Servier, Zentiva. He sits on advisory boards: Angelini, Celon (ended 2021), Douglas Pharmaceuticals, GH Research, Janssen, MSD, Novartis, Polpharma, Sanofi, Tasman Therapeutics. FUNDING This manuscript was funded by the ST-46 project (Medical University of Gdansk, Poland). Author Contribution Conceptualization: Magdalena Górska-Ponikowska. The first draft of the manuscript preparation including illustrations and tables: Zofia Winczewska. The review and editing: Zofia Winczewska, Magdalena Górska- Ponikowska, Wiesław Jerzy Cubała. Project management: Magdalena Górska-Ponikowska. Supervision: Magdalena Górska-Ponikowska. Funding acquisition: Magdalena Górska-Ponikowska.All authors reviewed and approved the final manuscript. Data Availability The data of this study are available from the corresponding author upon reasonable request. References McIntyre RS, Alsuwaidan M, Baune BT, Berk M, Demyttenaere K, Goldberg JF, Gorwood P, Ho R, Kasper S, Kennedy SH, Ly-Uson J, Mansur RB, McAllister-Williams RH, Murrough JW, Nemeroff CB, Nierenberg AA, Rosenblat JD, Sanacora G, Schatzberg AF, Shelton R, Stahl SM, Trivedi MH, Vieta E, Vinberg M, Williams N, Young AH, Maj M (2023) Treatment-resistant depression: definition, prevalence, detection, management, and investigational interventions. World Psychiatry 22(3):394–412. 10.1002/wps.21120 PMID: 37713549; PMCID: PMC10503923 Gałecki P, Talarowska M (2018) Inflammatory theory of depression. Psychiatr Pol 52(3):437–447 Ait Tayeb AEK, Poinsignon V, Chappell K, Bouligand J, Becquemont L, Verstuyft C (2023) Major Depressive Disorder and Oxidative Stress: A Review of Peripheral and Genetic Biomarkers According to Clinical Characteristics and Disease Stages. Antioxidants 12:942. https://doi.org/10.3390/antiox12040942 Cumurcu BE, Ozyurt H, Etikan I, Demir S, Karlidag R (2009) Total antioxidant capacity and total oxidant status in patients with major depression: impact of antidepressant treatment: TAC and TOS in major depression, Psychiatr. Clin Neurosci 63:639–645 Winczewska Z, Mechlińska A, Radziwiłłowicz P, Konieczna L, Drzeżdżon J, Jacewicz D, Wiglusz M, Bączek T, Cubała WJ, Górska-Ponikowska M (2025) Estrogen metabolites and hydrogen peroxide - Missing elements in the pathophysiology and possible treatment of treatment-resistant depression? Redox Biol 81:103547. 10.1016/j.redox.2025.103547 Epub 2025 Feb 20. PMID: 40068329; PMCID: PMC11938146 Kawczak P, Feszak I, Bączek T, Ketamine (2024) Esketamine, and Arketamine: Their Mechanisms of Action and Applications in the Treatment of Depression and Alleviation of Depressive Symptoms. Biomedicines 12(10):2283. 10.3390/biomedicines12102283 PMID: 39457596; PMCID: PMC11505277 Ivanović A, Petrović J, Stanić D, Nedeljković J, Ilić M, Jukić MM, Pejušković B, Pešić V (2025) Single subanesthetic dose of ketamine exerts antioxidant and antidepressive-like effect in ACTH-induced preclinical model of depression. Mol Cell Neurosci 133:104006 Epub 2025 Mar 27. PMID: 40157469 Bao H, Wang C, Xue X, Hu B, Guo Q (2024) CB1 receptor mediates anesthetic drug ketamine-induced neuroprotection against glutamate in HT22 cells. Exp Ther Med 27(6):268. 10.3892/etm.2024.12556 PMID: 38756904; PMCID: PMC11097274 Liang J, Wu S, Xie W, He H (2018) Ketamine ameliorates oxidative stress-induced apoptosis in experimental traumatic brain injury via the Nrf2 pathway. Drug Des Devel Ther 12:845–853. https://doi.org/10.2147/DDDT.S160046 de Carvalho Cartágenes S, Fernandes LMP, Carvalheiro TCVS, de Sousa TM, Gomes ARQ, Monteiro MC, de Oliveira Paraense RS, Crespo-López ME, Lima RR, Fontes-Júnior EA, Prediger RD, Maia CSF (2019) Special K Drug on Adolescent Rats: Oxidative Damage and Neurobehavioral Impairments. Oxid Med Cell Longev. ; 2019:5452727. 10.1155/2019/5452727 . PMID: 31001375; PMCID: PMC6437740 Medeiros GC, Demo I, Goes FS, Zarate CA Jr, Gould TD (2024) Personalized use of ketamine and esketamine for treatment-resistant depression. Transl Psychiatry 14(1):481. 10.1038/s41398-024-03180-8 PMID: 39613748; PMCID: PMC11607365 Weckmann K, Deery MJ, Howard JA Ketamine’s antidepressant effect is mediated by energy metabolism and antioxidant defense system. et al (2017) https://doi.org/10.1038/s41598 -017-16183-x [13] Kajumba MM, Kakooza-Mwesige A, Nakasujja N, Koltai D, Canli T. Treatment-resistant depression: molecular mechanisms and management. Mol Biomed. 2024;5(1):43. doi: 10.1186/s43556-024-00205-y. PMID: 39414710; PMCID: PMC11485009 Zhang M, Lyu D, Wang F, Shi S, Wang M, Yang W, Huang H, Wei Z, Chen S, Xu Y, Hong W. Ketamine May Exert Rapid Antidepressant Effects Through Modulation of Neuroplasticity, Autophagy, and Ferroptosis in the Habenular Nucleus. Neuroscience. ;506:29–37. doi:, Zhang Y, Cai Q, Wang L, Zhang B (2022) The Impact of Esketamine on Depression: Targeting Oxidative Stress and Neuronal Apoptosis Through BDNF/TrkB/PI3K/AKT Pathway Activation. Neuropsychiatr Dis Treat . 2025;21:1783–1793 https://doi.org/10.2147/NDT.S502090 Additional Declarations Competing interest reported. Zofia Winczewska, Magdalena Górska-Ponikowska have no conflict of interests. Wiesław Jerzy Cubała has received grants: Acadia, Alkermes, Allergan, Angelini, Auspex Pharmaceuticals, Beckley Psytech, BMS, Celon, Cephalon, Compass Pathways, Cortexyme, Ferrier, Forest Laboratories, GedeonRichter, GH Research, GWPharmaceuticals, HMNC Brain Health, IntraCellular Therapies, Janssen, KCR, Lilly, Lundbeck, MindMed, Minerva, MSD, NIH, Neumora, Novartis, Orion, Otsuka, Recognify Life Sciences, Sanofi, Seaport, Servier. He has received honoraria: Adamed, Angelini, AstraZeneca, BMS, Celon, GH Research, GSK, Janssen, KRKA, Lekam, Lundbeck, Minerva, NeuroCog, Novartis, Orion, Pfizer, Polfa Tarchomin, Sanofi, Servier, Zentiva. He sits on advisory boards: Angelini, Celon (ended 2021), Douglas Pharmaceuticals, GH Research, Janssen, MSD, Novartis, Polpharma, Sanofi, Tasman Therapeutics. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8624006","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":592379769,"identity":"6e3918fe-011d-4094-84f8-1e7fe53ff619","order_by":0,"name":"Zofia Winczewska","email":"","orcid":"","institution":"Medical University of Gdansk","correspondingAuthor":false,"prefix":"","firstName":"Zofia","middleName":"","lastName":"Winczewska","suffix":""},{"id":592379771,"identity":"eb38d4f8-714f-4f18-8e14-c09cf3033e66","order_by":1,"name":"Magdalena Górska-Ponikowska","email":"data:image/png;base64,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","orcid":"","institution":"Medical University of Gdansk","correspondingAuthor":true,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Górska-Ponikowska","suffix":""},{"id":592379772,"identity":"dbb454f5-84a0-4181-b22e-e3aa20b823f4","order_by":2,"name":"Wiesław Jerzy Cubała","email":"","orcid":"","institution":"Medical University of Gdansk","correspondingAuthor":false,"prefix":"","firstName":"Wiesław","middleName":"Jerzy","lastName":"Cubała","suffix":""}],"badges":[],"createdAt":"2026-01-17 06:53:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8624006/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8624006/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102849355,"identity":"6f36a990-5dcd-456b-97bd-2827c596ce2a","added_by":"auto","created_at":"2026-02-17 13:56:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViability HT22 cells after 24h incubation with H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and ketamine: 25 ng/ml ketamine and H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e in a concentration range of 10 µM – 1000 µM were added and incubated for 24h. Cells were harvested and viability was determined by MTT assay. * p \u0026lt;0.1, *** p \u0026lt;0.001, **** p \u0026lt;0.0001 vs control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8624006/v1/6ab8e83e9ee59b9edcc9335e.png"},{"id":103528233,"identity":"1d33a4e7-d7d3-45ae-b4e0-ecb172cb85b0","added_by":"auto","created_at":"2026-02-26 16:25:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":570182,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8624006/v1/f1deaae9-cbff-46ec-92c6-1a6b8e2de4a8.pdf"}],"financialInterests":"Competing interest reported. Zofia Winczewska, Magdalena Górska-Ponikowska have no conflict of interests. \nWiesław Jerzy Cubała has received grants: Acadia, Alkermes, Allergan, Angelini, Auspex Pharmaceuticals, Beckley Psytech, BMS, Celon, Cephalon, Compass Pathways, Cortexyme, Ferrier, Forest Laboratories, GedeonRichter, GH Research, GWPharmaceuticals, HMNC Brain Health, IntraCellular Therapies, Janssen, KCR, Lilly, Lundbeck, MindMed, Minerva, MSD, NIH, Neumora, Novartis, Orion, Otsuka, Recognify Life Sciences, Sanofi, Seaport, Servier. \nHe has received honoraria: Adamed, Angelini, AstraZeneca, BMS, Celon, GH Research, GSK, Janssen, KRKA, Lekam, Lundbeck, Minerva, NeuroCog, Novartis, Orion, Pfizer, Polfa Tarchomin, Sanofi, Servier, Zentiva. He sits on advisory boards: Angelini, Celon (ended 2021), Douglas Pharmaceuticals, GH Research, Janssen, MSD, Novartis, Polpharma, Sanofi, Tasman Therapeutics.","formattedTitle":"The Antioxidant Activity of Ketamine: Threshold-Dependent Mechanism in Treatment-Resistant Depression?","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTRD is a severe subtype of major depressive disorder (MDD), occurring in approximately 30% of patients who do not achieve clinical improvement despite multiple pharmacological interventions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The lack of efficacy of antidepressants in a significant percentage of patients indicates that the mechanisms associated with symptom persistence in TRD go beyond the monoamine theory [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The role of OS in the etiopathogenesis of depression is particularly interesting and well established. One of the main mediators of OS is H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e - in people with severe, recurrent depression, elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations are observed along with depletion of antioxidant defenses, including low levels of superoxide dismutase (SOD) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations are characteristic of individuals with recurrent, severe depression. A decrease in total antioxidant capacity (TAC) is observed, accompanied by an increase in total oxidation status (TOS) and markers of inflammation, including 8-OHdG [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The results of a recently published study by our team also showed high levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in individuals with TRD, the source of which was addressed in abnormal estrogen metabolism inducing OS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In view of the above data OS is positioned as an important factor in the development of TRD and responsible for the heterogeneity of the disease, which may be behind the different responses of patients to treatment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKetamine, classified as a rapid-acting antidepressant (RAAD) used in the treatment of TRD, produces a response in 50\u0026ndash;70% of patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although the mechanism of action of ketamine involves non-competitive antagonism of the N-methyl-D-aspartate (NMDAR) receptor, its antidepressant effect may be at least partly due to its antioxidant activity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Preclinical studies have shown that ketamine administered under conditions of stress induced by repeated ACTH administration developed an antidepressant effect through an antioxidant mechanism - a decrease in TOS and malondialdehyde (MDA) was observed, with a simultaneous increase in superoxide dismutase (SOD) and paraoxonase 1 (PON-1) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ketamine also reduced oxidative damage in the HT22 cell model treated with glutamate, which induced OS in depression. An improvement in antioxidant status and neuroprotective effects via the cannabinoid receptor (CBD-1) were noted [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interestingly, in models of traumatic brain injury, ketamine also showed a protective effect by reducing OS, including a decrease in MDA, an increase in SOD and glutathione peroxidase (GPx), and increased expression of antioxidant factors associated with the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, there are studies with contradictory results, which show that under stress-free conditions, ketamine led to an increase in MDA levels and thus a pro-inflammatory effect [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Another study also refers to the dual nature of ketamine, whose neuroprotective effect may be conditional and dependent on circumstances, including the dose of ketamine used [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The scientific literature confirms a varied response to ketamine in patients, which highlights the urgent need to individualize indications by identifying predictors of effective response to treatment [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eExperiments employed immortalized mouse hippocampal cell line HT22 (kindly gifted by Tilman Grune from Institute of Nutrition, Department of Nutritional Toxicology, Friedrich Schiller University (Jena, Germany) and by Kelvin Davies from University of Southern California (USC, Los Angeles, CA, USA)). The cells were grown in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s low glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Treatment\u003c/h3\u003e\n\u003cp\u003eHT22 cells were seeded in culture media at appropriate density 24 h before treatment. Next, the cells were treated with 25 ng/ml ketamine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in a concentration range of 10 \u0026micro;M \u0026ndash; 1000 \u0026micro;M. The ketamine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solutions were prepared in sterile water.\u003c/p\u003e\n\u003ch3\u003eCell Viability/Cell Proliferation Assay (MTT Assay)\u003c/h3\u003e\n\u003cp\u003e10,000 HT22 cells per well were seeded in 96-well plates. After 24 hours, the cells were treated with 25 ng/ml ketamine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in a concentration range of 10 \u0026micro;M \u0026ndash; 1000 \u0026micro;M for the next 24h. Solvent-treated control cells were regarded as 100% viable. MTT solution (Sigma-Aldrich, Poland) at a concentration of 0.5 mg/mL was used following incubation. The plates were centrifuged (700 g for 10 min) to extract the supernatant after 4 hours at 37\u0026deg;C. 100 \u0026micro;l of DMSO (Sigma-Aldrich, Poland) were used to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader (BioTek Instruments, Inc., USA). Percentage of the control was used to display the data. Each sample was prepared in three repetitions.\u003c/p\u003e\n\u003ch3\u003eStatistical analysis of the results\u003c/h3\u003e\n\u003cp\u003eThe mean and standard deviation (SD) derived from three samples are used to present the results. The Student's t-test was used to evaluate the differences between the control samples and the ketamine-treated samples and to compare H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated samples to the one treated with the mixture of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and ketamine. Statistical significance was established at a p-value of 0.05. Data analysis was done using GraphPad Prism (GraphPad Software, Inc., version 8, USA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of ketamine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on cell viability\u003c/h2\u003e \u003cp\u003eIn order to evaluate the impact of ketamine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on HT22 cells, a cytotoxicity MTT assay was carried out. Precisely, HTT cell viability was determined after incubating the cells for 24 hours with 25 ng/ml ketamine and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in a concentration range of 10 \u0026micro;M \u0026ndash; 1000 \u0026micro;M. The sample's percentage of viable cells was determined in relation to the 100% viable control cells.\u003c/p\u003e \u003cp\u003eHT22 cell viability was as follows: 34% (\u0026plusmn;\u0026thinsp;4.78%) after treatment with 25 ng/ml ketamine, 86% (\u0026plusmn;\u0026thinsp;3.66%) 10 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, whereas 31% (\u0026plusmn;\u0026thinsp;0.85%) after incubation with the mixture 10 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine. 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decreased HT22 cell viability to 68% (\u0026plusmn;\u0026thinsp;3.26%), 30 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine 34% (\u0026plusmn;\u0026thinsp;6.87%), 50 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 63% (\u0026plusmn;\u0026thinsp;3.45%), 50 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine 35% (\u0026plusmn;\u0026thinsp;3.66%), 125 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 65% (\u0026plusmn;\u0026thinsp;10.78%), 125 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine 36% (\u0026plusmn;\u0026thinsp;5.04%), 250 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 87% (\u0026plusmn;\u0026thinsp;6.03%), 250 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine 40% (\u0026plusmn;\u0026thinsp;8.48%), 500 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 73% (\u0026plusmn;\u0026thinsp;4.62%), 500 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine 39% (\u0026plusmn;\u0026thinsp;5.23%), 750 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 44% (\u0026plusmn;\u0026thinsp;1.46%), 750 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine 31% (\u0026plusmn;\u0026thinsp;3.66%). In case of 1000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e there were 12% (\u0026plusmn;\u0026thinsp;1.63%) viable, however after incubation with 1000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine cell viability was 38% (\u0026plusmn;\u0026thinsp;9.12%). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe results presented in this study are consistent with the scientific literature, which indicates that the antioxidant activity of ketamine may mediate its antidepressant effects. However in this study, we expanded the context of ketamine's antioxidant activity to include its potential threshold effect depending on the severity of depression, as the cytoprotective effect only became apparent at high concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Previous studies have consistently pointed to the antidepressant effect of ketamine through the modulation of signaling pathways associated with OS, including glutathione (GSH), Nrf2, SOD, and PON-1 [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It has also been suggested that the effectiveness of ketamine in people with TRD may be due to its beneficial effect on mitochondrial energy metabolism, whose dysfunction is associated with the development and progression of depression [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In a study by Weckmann et al., it was noted that after treatment with ketamine, the activation of the mTOR pathway increased, which led to an increase in the expression of genes related to energy metabolism and the promotion of mitochondrial biogenesis and redox balance regulation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The high efficacy of ketamine treatment in patients with TRD may therefore result from the drug's beneficial effect on mitochondrial energy metabolism, which is closely linked to the glutamatergic system, whose increased activity plays a key role in the progression of depressive disorders [13]. In another study, the antidepressant effect of ketamine was linked to the induction of autophagy, inhibition of ferroptosis, and improvement of neuroplasticity, i.e., pathways closely related to OS and the persistence of depressive symptoms [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, it has been suggested that ketamine may act differently in patients depending on specific subtypes of depression. One study indicated that ketamine was not effective in people with mild depression, suggesting that the effect of ketamine is modulated by the severity of the disease [15].\u003c/p\u003e \u003cp\u003eThis study discusses a new, previously undescribed protective effect of ketamine on HT22 neuronal cells in which OS was induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Importantly, a threshold, conditional protective effect of ketamine was observed not at every level of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exposure, but at specific concentrations corresponding to critical OS overload. These observations therefore suggest the existence of a certain ceiling in high ranges of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations at which the antioxidant mechanisms of ketamine are activated. Consistent with these results are observations of the opposite, pro-oxidative effect of ketamine under low stress conditions, suggesting variability in response to ketamine treatment potentially dependent on the biological context. These results deepen our understanding of the antioxidant mechanisms of ketamine and correspond with clinical observations suggesting that the effect of ketamine depends on the phenotype of depression.\u003c/p\u003e"},{"header":"CONCLUSIONS AND FUTURE DIRECTIONS","content":"\u003cp\u003eNumerous studies indicate that OS may influence the heterogeneity of depression and thus the varying response to treatment. Therefore, more and more studies are focusing on the search for biomarkers that would allow for targeted and effective treatment of TRD. The results presented in this study indicated that the cytoprotective effect of ketamine was only evident at high concentrations of H₂O₂, suggesting the existence of a potential threshold for the activation of antioxidant mechanisms and possibly explaining the varying response to treatment depending on biological background. These observations provide a promising basis for further research into the mechanisms of action of ketamine, as well as for clinical trials to assess whether the efficacy of ketamine correlates with the severity of OS and whether patients with higher H₂O₂ levels are indeed more likely to respond to treatment. This highlights the potential of H₂O₂ as a predictor of response to treatment in TRD, which may contribute to the personalization of therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST STATEMENT\u003c/h2\u003e \u003cp\u003eWiesław Jerzy Cubała has received grants: Acadia, Alkermes, Allergan, Angelini, Auspex Pharmaceuticals, Beckley Psytech, BMS, Celon, Cephalon, Compass Pathways, Cortexyme, Ferrier, Forest Laboratories, GedeonRichter, GH Research, GWPharmaceuticals, HMNC Brain Health, IntraCellular Therapies, Janssen, KCR, Lilly, Lundbeck, MindMed, Minerva, MSD, NIH, Neumora, Novartis, Orion, Otsuka, Recognify Life Sciences, Sanofi, Seaport, Servier.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eZofia Winczewska, Magdalena G\u0026oacute;rska-Ponikowska have no conflict of interests. Wiesław Jerzy Cubała has received grants: Acadia, Alkermes, Allergan, Angelini, Auspex Pharmaceuticals, Beckley Psytech, BMS, Celon, Cephalon, Compass Pathways, Cortexyme, Ferrier, Forest Laboratories, GedeonRichter, GH Research, GWPharmaceuticals, HMNC Brain Health, IntraCellular Therapies, Janssen, KCR, Lilly, Lundbeck, MindMed, Minerva, MSD, NIH, Neumora, Novartis, Orion, Otsuka, Recognify Life Sciences, Sanofi, Seaport, Servier. He has received honoraria: Adamed, Angelini, AstraZeneca, BMS, Celon, GH Research, GSK, Janssen, KRKA, Lekam, Lundbeck, Minerva, NeuroCog, Novartis, Orion, Pfizer, Polfa Tarchomin, Sanofi, Servier, Zentiva. He sits on advisory boards: Angelini, Celon (ended 2021), Douglas Pharmaceuticals, GH Research, Janssen, MSD, Novartis, Polpharma, Sanofi, Tasman Therapeutics.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis manuscript was funded by the ST-46 project (Medical University of Gdansk, Poland).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Magdalena G\u0026oacute;rska-Ponikowska. The first draft of the manuscript preparation including illustrations and tables: Zofia Winczewska. The review and editing: Zofia Winczewska, Magdalena G\u0026oacute;rska- Ponikowska, Wiesław Jerzy Cubała. Project management: Magdalena G\u0026oacute;rska-Ponikowska. Supervision: Magdalena G\u0026oacute;rska-Ponikowska. Funding acquisition: Magdalena G\u0026oacute;rska-Ponikowska.All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcIntyre RS, Alsuwaidan M, Baune BT, Berk M, Demyttenaere K, Goldberg JF, Gorwood P, Ho R, Kasper S, Kennedy SH, Ly-Uson J, Mansur RB, McAllister-Williams RH, Murrough JW, Nemeroff CB, Nierenberg AA, Rosenblat JD, Sanacora G, Schatzberg AF, Shelton R, Stahl SM, Trivedi MH, Vieta E, Vinberg M, Williams N, Young AH, Maj M (2023) Treatment-resistant depression: definition, prevalence, detection, management, and investigational interventions. World Psychiatry 22(3):394\u0026ndash;412. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/wps.21120\u003c/span\u003e\u003cspan address=\"10.1002/wps.21120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 37713549; PMCID: PMC10503923\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGałecki P, Talarowska M (2018) Inflammatory theory of depression. Psychiatr Pol 52(3):437\u0026ndash;447\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAit Tayeb AEK, Poinsignon V, Chappell K, Bouligand J, Becquemont L, Verstuyft C (2023) Major Depressive Disorder and Oxidative Stress: A Review of Peripheral and Genetic Biomarkers According to Clinical Characteristics and Disease Stages. Antioxidants 12:942. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox12040942\u003c/span\u003e\u003cspan address=\"10.3390/antiox12040942\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCumurcu BE, Ozyurt H, Etikan I, Demir S, Karlidag R (2009) Total antioxidant capacity and total oxidant status in patients with major depression: impact of antidepressant treatment: TAC and TOS in major depression, Psychiatr. Clin Neurosci 63:639\u0026ndash;645\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWinczewska Z, Mechlińska A, Radziwiłłowicz P, Konieczna L, Drzeżdżon J, Jacewicz D, Wiglusz M, Bączek T, Cubała WJ, G\u0026oacute;rska-Ponikowska M (2025) Estrogen metabolites and hydrogen peroxide - Missing elements in the pathophysiology and possible treatment of treatment-resistant depression? Redox Biol 81:103547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.redox.2025.103547\u003c/span\u003e\u003cspan address=\"10.1016/j.redox.2025.103547\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eEpub 2025 Feb 20. PMID: 40068329; PMCID: PMC11938146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawczak P, Feszak I, Bączek T, Ketamine (2024) Esketamine, and Arketamine: Their Mechanisms of Action and Applications in the Treatment of Depression and Alleviation of Depressive Symptoms. Biomedicines 12(10):2283. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biomedicines12102283\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines12102283\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 39457596; PMCID: PMC11505277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIvanović A, Petrović J, Stanić D, Nedeljković J, Ilić M, Jukić MM, Pejušković B, Pešić V (2025) Single subanesthetic dose of ketamine exerts antioxidant and antidepressive-like effect in ACTH-induced preclinical model of depression. Mol Cell Neurosci 133:104006 Epub 2025 Mar 27. PMID: 40157469\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao H, Wang C, Xue X, Hu B, Guo Q (2024) CB1 receptor mediates anesthetic drug ketamine-induced neuroprotection against glutamate in HT22 cells. Exp Ther Med 27(6):268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/etm.2024.12556\u003c/span\u003e\u003cspan address=\"10.3892/etm.2024.12556\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 38756904; PMCID: PMC11097274\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang J, Wu S, Xie W, He H (2018) Ketamine ameliorates oxidative stress-induced apoptosis in experimental traumatic brain injury via the Nrf2 pathway. Drug Des Devel Ther 12:845\u0026ndash;853. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/DDDT.S160046\u003c/span\u003e\u003cspan address=\"10.2147/DDDT.S160046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Carvalho Cart\u0026aacute;genes S, Fernandes LMP, Carvalheiro TCVS, de Sousa TM, Gomes ARQ, Monteiro MC, de Oliveira Paraense RS, Crespo-L\u0026oacute;pez ME, Lima RR, Fontes-J\u0026uacute;nior EA, Prediger RD, Maia CSF (2019) Special K Drug on Adolescent Rats: Oxidative Damage and Neurobehavioral Impairments. Oxid Med Cell Longev. ; 2019:5452727. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2019/5452727\u003c/span\u003e\u003cspan address=\"10.1155/2019/5452727\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 31001375; PMCID: PMC6437740\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedeiros GC, Demo I, Goes FS, Zarate CA Jr, Gould TD (2024) Personalized use of ketamine and esketamine for treatment-resistant depression. Transl Psychiatry 14(1):481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41398-024-03180-8\u003c/span\u003e\u003cspan address=\"10.1038/s41398-024-03180-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 39613748; PMCID: PMC11607365\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeckmann K, Deery MJ, Howard JA Ketamine\u0026rsquo;s antidepressant effect is mediated by energy metabolism and antioxidant defense system. et al (2017) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598\u003c/span\u003e\u003cspan address=\"10.1038/s41598\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e-017-16183-x [13] Kajumba MM, Kakooza-Mwesige A, Nakasujja N, Koltai D, Canli T. Treatment-resistant depression: molecular mechanisms and management. Mol Biomed. 2024;5(1):43. doi: 10.1186/s43556-024-00205-y. PMID: 39414710; PMCID: PMC11485009\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang M, Lyu D, Wang F, Shi S, Wang M, Yang W, Huang H, Wei Z, Chen S, Xu Y, Hong W. Ketamine May Exert Rapid Antidepressant Effects Through Modulation of Neuroplasticity, Autophagy, and Ferroptosis in the Habenular Nucleus. Neuroscience. ;506:29\u0026ndash;37. doi:, Zhang Y, Cai Q, Wang L, Zhang B (2022) The Impact of Esketamine on Depression: Targeting Oxidative Stress and Neuronal Apoptosis Through BDNF/TrkB/PI3K/AKT Pathway Activation. \u003cem\u003eNeuropsychiatr Dis Treat\u003c/em\u003e. 2025;21:1783\u0026ndash;1793 https://doi.org/10.2147/NDT.S502090\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"treatment resistant depression, ketamine, oxidative stress, hydrogen peroxide, HT22","lastPublishedDoi":"10.21203/rs.3.rs-8624006/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8624006/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim of study:\u003c/h2\u003e \u003cp\u003eThe antidepressant effect of ketamine may be at least partly due to its antioxidant activity. The aim of the study was to assess whether ketamine develops a protective effect on HT22 neuronal cells under conditions of oxidative stress (OS) induced by hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\u003ch2\u003eMaterial and methods\u003c/h2\u003e \u003cp\u003eThe experiment was performed on an immortalized mouse hippocampal HT22 cell line. Cell viability after treatment with 25 ng/ml ketamine and 10 \u0026micro;M \u0026ndash; 1000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24h was assessed by MTT assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eKetamine at a concentration of 25 ng/mL increased the viability of HT22 neuronal cells under conditions of OS mimicked by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, but only for a concentration of 1000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which reflects the high level of OS typical of treatment-resistant depression (TRD). At 1000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, cell viability was 12% (\u0026plusmn;\u0026thinsp;1.63%), while after incubation with 1000 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;25 ng/ml ketamine, cell viability was 38% (\u0026plusmn;\u0026thinsp;9.12%). The results suggest that ketamine has a protective effect on HT22 cells, but that this effect is nonlinear and dependent on OS intensity, activated only at critical H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e overload.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePreliminary results suggest that ketamine reverses the cytotoxicity of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e on HT22 cells, but only at specific, high concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e typical of severe subtypes of depression. This indicates a threshold, antioxidant mechanism of ketamine's antidepressant action, which could be used in further research on predictive models of response to ketamine treatment, which in the future could translate into individualized treatment and increased therapeutic success.\u003c/p\u003e","manuscriptTitle":"The Antioxidant Activity of Ketamine: Threshold-Dependent Mechanism in Treatment-Resistant Depression?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 13:56:03","doi":"10.21203/rs.3.rs-8624006/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d67abd73-3a12-4436-9132-48b8df7573f0","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-26T16:25:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 13:56:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8624006","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8624006","identity":"rs-8624006","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}