{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T22:21:26Z","timestamp":1768947686499,"version":"3.49.0"},"reference-count":40,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T00:00:00Z","timestamp":1768608000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Education, Science and Sport of the Republic of Lithuania via Research Council of Lithuania","award":["S-A-UEI-23-7"],"award-info":[{"award-number":["S-A-UEI-23-7"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Medicina"],"abstract":"<jats:p>Background and Objectives: SARS-CoV-2 produces potentially pathogenic molecules, such as single-stranded RNA and spike proteins, which can potentially activate microglial cells. In this study, we aimed to investigate whether SARS-CoV-2 spike protein S1 and mRNA vaccines can cause neurotoxicity directly or through microglial involvement. Materials and Methods: Primary cerebellar granule cell cultures isolated from Wistar rats and organotypic hippocampal slice cultures from transgenic C57BL\/6J mice were used in the experiments. Imaging and quantitative analysis of cell viability, proliferation, and phagocytic activity were performed using light and fluorescence microscopy. Results: The exogenous SARS-CoV-2 S1 protein at 50 \u00b5g\/mL concentration induced neuronal cell death in neuronal\u2013glial co-cultures and stimulated microglial proliferation during the first 3 days of exposure without an effect on inflammatory cytokine secretion. Single application of Tozinameran\/Riltozinameran and Original\/Omicron BA. 4\u20135 vaccines did not affect neuronal viability and total neuronal number in cell co-cultures after 7 days of exposure. In contrast, three repeated treatments with mRNA vaccines at 6 ng\/mL caused microglial proliferation without affecting microglial phagocytosis and TNF-\u03b1 release. In organotypic brain slice cultures, only Tozinameran\/Riltozinameran stimulated microglial cell proliferation in female brain slices, while male brain slices remained unaffected by both vaccines, indicating sex-dependent effects. Conclusions: The findings suggest that mRNA vaccines do not exert neurotoxic effects in primary neuronal\u2013glial co-cultures, but induce microglial proliferation, particularly in female brains in the absence of inflammatory cytokine release. SARS-CoV-2 S1 protein at high concentrations directly induces neuronal death.<\/jats:p>","DOI":"10.3390\/medicina62010198","type":"journal-article","created":{"date-parts":[[2026,1,19]],"date-time":"2026-01-19T08:23:56Z","timestamp":1768811036000},"page":"198","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Effects of Exogenous SARS-CoV-2 S1 Protein and mRNA Vaccines on Mixed Neuronal\u2013Glial Cell Cultures"],"prefix":"10.3390","volume":"62","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-2692-524X","authenticated-orcid":false,"given":"Vytenis","family":"Markevi\u010dius","sequence":"first","affiliation":[{"name":"Neuroscience Institute, Lithuanian University of Health Sciences, Eiveniu Str. 4, LT-50161 Kaunas, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4881-5754","authenticated-orcid":false,"given":"Eimina","family":"Dirvelyt\u0117-Valausk\u0117","sequence":"additional","affiliation":[{"name":"Vilnius University Life Sciences Center-European Molecular Biology Laboratory (VU LSC-EMBL) Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Sauletekio Str. 7, LT-10257 Vilnius, Lithuania"},{"name":"Institute of Bioscience, Life Sciences Center, Vilnius University, Sauletekio Str. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Urt\u0117","family":"Neni\u0161kyt\u0117","sequence":"additional","affiliation":[{"name":"Vilnius University Life Sciences Center-European Molecular Biology Laboratory (VU LSC-EMBL) Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, Sauletekio Str. 7, LT-10257 Vilnius, Lithuania"},{"name":"Institute of Bioscience, Life Sciences Center, Vilnius University, Sauletekio Str. 7, LT-10257 Vilnius, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vilmant\u0117","family":"Borutait\u0117","sequence":"additional","affiliation":[{"name":"Neuroscience Institute, Lithuanian University of Health Sciences, Eiveniu Str. 4, LT-50161 Kaunas, Lithuania"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1038\/s41591-020-0968-3","article-title":"Extrapulmonary Manifestations of COVID-19","volume":"26","author":"Gupta","year":"2020","journal-title":"Nat. Med."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Reza-Zald\u00edvar, E.E., Hern\u00e1ndez-Sapi\u00e9ns, M.A., Minjarez, B., G\u00f3mez-Pinedo, U., M\u00e1rquez-Aguirre, A.L., Mateos-D\u00edaz, J.C., Matias-Guiu, J., and Canales-Aguirre, A.A. (2021). Infection Mechanism of SARS-CoV-2 and Its Implication on the Nervous System. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.621735"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1111\/jnc.15736","article-title":"SARS-CoV-2, Aging, and Post-COVID-19 Neurodegeneration","volume":"165","author":"Strong","year":"2022","journal-title":"J. Neurochem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1594","DOI":"10.1016\/j.immuni.2021.06.002","article-title":"Deep Spatial Profiling of Human COVID-19 Brains Reveals Neuroinflammation with Distinct Microanatomical Microglia-T-Cell Interactions","volume":"54","author":"Schwabenland","year":"2021","journal-title":"Immunity"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.bbi.2021.12.007","article-title":"SARS-CoV-2 Spike S1 Subunit Induces Neuroinflammatory, Microglial and Behavioral Sickness Responses: Evidence of PAMP-like Properties","volume":"100","author":"Frank","year":"2022","journal-title":"Brain Behav. Immun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e0151121","DOI":"10.1128\/JVI.01511-21","article-title":"SARS-CoV-2 Causes Lung Infection without Severe Disease in Human ACE2 Knock-In Mice","volume":"96","author":"Winkler","year":"2022","journal-title":"J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e01091-22","DOI":"10.1128\/spectrum.01091-22","article-title":"SARS-CoV-2 Infection of Microglia Elicits Proinflammatory Activation and Apoptotic Cell Death","volume":"10","author":"Jeong","year":"2022","journal-title":"Microbiol. Spectr."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1007\/s12035-021-02593-6","article-title":"SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia","volume":"59","author":"Olajide","year":"2022","journal-title":"Mol. Neurobiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1007\/s11481-021-10015-6","article-title":"Mitochondrial Dynamics in SARS-COV2 Spike Protein Treated Human Microglia: Implications for Neuro-COVID","volume":"16","author":"Clough","year":"2021","journal-title":"J. Neuroimmune Pharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"100112","DOI":"10.1016\/j.crneur.2023.100112","article-title":"The SARS-CoV-2 Spike Glycoprotein Interacts with MAO-B and Impairs Mitochondrial Energetics","volume":"5","author":"Pileggi","year":"2023","journal-title":"Curr. Res. Neurobiol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Alves, V.S., Santos, S.A.C.S., Leite-Aguiar, R., Paiva-Pereira, E., Dos Reis, R.R., Calazans, M.L., Fernandes, G.G., Ant\u00f4nio, L.S., de Lima, E.V., and Kurtenbach, E. (2023). SARS-CoV-2 Spike Protein Alters Microglial Purinergic Signaling. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1158460"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"115020","DOI":"10.1016\/j.expneurol.2024.115020","article-title":"SARS-CoV-2 Spike S1 Protein Induces Microglial NLRP3-Dependent Neuroinflammation and Cognitive Impairment in Mice","volume":"383","author":"Jiang","year":"2025","journal-title":"Exp. Neurol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2814","DOI":"10.1167\/iovs.15-18509","article-title":"Differentiated Expression Patterns and Phagocytic Activities of Type 1 and 2 Microglia","volume":"57","author":"Haga","year":"2016","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1111\/jnc.15327","article-title":"Microglial Phagocytosis of Neurons in Neurodegeneration, and Its Regulation","volume":"158","author":"Butler","year":"2021","journal-title":"J. Neurochem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"817","DOI":"10.1038\/s41573-021-00283-5","article-title":"MRNA Vaccines for Infectious Diseases: Principles, Delivery and Clinical Translation","volume":"20","author":"Chaudhary","year":"2021","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1080\/14760584.2024.2397026","article-title":"A Review of the Immunogenicity and Safety of Booster Doses of Omicron Variant-Containing MRNA-1273 COVID-19 Vaccines in Adults and Children","volume":"23","author":"Priddy","year":"2024","journal-title":"Expert Rev. Vaccin."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1056\/NEJMoa2035389","article-title":"Efficacy and Safety of the MRNA-1273 SARS-CoV-2 Vaccine","volume":"384","author":"Baden","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1111\/imj.15623","article-title":"Pfizer\/BioNtech BNT162b2: Adverse Events and Insights from an Australian Mass Vaccination Clinic for COVID-19","volume":"52","author":"Halder","year":"2022","journal-title":"Intern. Med. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1038\/s41591-022-02092-8","article-title":"Bivalent SARS-CoV-2 MRNA Vaccines Increase Breadth of Neutralization and Protect against the BA.5 Omicron Variant in Mice","volume":"29","author":"Scheaffer","year":"2022","journal-title":"Nat. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2321994","DOI":"10.1080\/22221751.2024.2321994","article-title":"Effectiveness of a Broad-Spectrum Bivalent MRNA Vaccine against SARS-CoV-2 Variants in Preclinical Studies","volume":"13","author":"Lu","year":"2024","journal-title":"Emerg. Microbes Infect."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1897","DOI":"10.1016\/j.ymthe.2022.01.001","article-title":"A Self-Amplifying MRNA SARS-CoV-2 Vaccine Candidate Induces Safe and Robust Protective Immunity in Preclinical Models","volume":"30","author":"Maruggi","year":"2022","journal-title":"Mol. Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"114204","DOI":"10.1016\/j.ejpb.2024.114204","article-title":"Less Is More: Self-Amplifying MRNA Becomes Self-Killing upon Dose Escalation in Immune-Competent Retinal Cells","volume":"196","author":"Vanluchene","year":"2024","journal-title":"Eur. J. Pharm. Biopharm."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"NEJMoa2203315","DOI":"10.1056\/NEJMoa2203315","article-title":"Evaluation of MRNA-1273 COVID-19 Vaccine in Children 6 to 11 Years of Age","volume":"386","author":"Creech","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.1056\/NEJMoa2109522","article-title":"Evaluation of MRNA-1273 SARS-CoV-2 Vaccine in Adolescents","volume":"385","author":"Ali","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1186\/s40001-023-00992-0","article-title":"A Review of Neurological Side Effects of COVID-19 Vaccination","volume":"28","author":"Hosseini","year":"2023","journal-title":"Eur. J. Med. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"239821282097980","DOI":"10.1177\/2398212820979802","article-title":"The Role of Cytokines in Modulating Learning and Memory and Brain Plasticity","volume":"4","author":"Bourgognon","year":"2020","journal-title":"Brain Neurosci. Adv."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e111790","DOI":"10.15252\/embj.2022111790","article-title":"Phospholipid Scramblase Xkr8 Is Required for Developmental Axon Pruning via Phosphatidylserine Exposure","volume":"42","author":"Neniskyte","year":"2023","journal-title":"EMBO J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6480","DOI":"10.1523\/JNEUROSCI.21-17-06480.2001","article-title":"Inflammatory Neurodegeneration Mediated by Nitric Oxide from Activated Glia-Inhibiting Neuronal Respiration, Causing Glutamate Release and Excitotoxicity","volume":"21","author":"Brown","year":"2001","journal-title":"J. Neurosci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10813","DOI":"10.1038\/s41598-023-37887-3","article-title":"Extracellular Tau Stimulates Phagocytosis of Living Neurons by Activated Microglia via Toll-like 4 Receptor\u2013NLRP3 Inflammasome\u2013Caspase-1 Signalling Axis","volume":"13","author":"Pampuscenko","year":"2023","journal-title":"Sci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Jankeviciute, S., Svirskiene, N., Svirskis, G., and Borutaite, V. (2021). Effects of Metformin on Spontaneous Ca2+ Signals in Cultured Microglia Cells under Normoxic and Hypoxic Conditions. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22179493"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/0165-0270(91)90128-M","article-title":"A Simple Method for Organotypic Cultures of Nervous Tissue","volume":"37","author":"Stoppini","year":"1991","journal-title":"J. Neurosci. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1235","DOI":"10.1038\/nprot.2012.058","article-title":"Assessment of Mitochondrial Respiratory Chain Enzymatic Activities on Tissues and Cultured Cells","volume":"7","author":"Spinazzi","year":"2012","journal-title":"Nat. Protoc."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7631","DOI":"10.1073\/pnas.95.13.7631","article-title":"Persistent Inhibition of Cell Respiration by Nitric Oxide: Crucial Role of S-Nitrosylation of Mitochondrial Complex I and Protective Action of Glutathione","volume":"95","author":"Clementi","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1228","DOI":"10.1038\/s41467-018-03566-5","article-title":"Microglia Remodel Synapses by Presynaptic Trogocytosis and Spine Head Filopodia Induction","volume":"9","author":"Weinhard","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Barreda, D., Santiago, C., Rodr\u00edguez, J.R., Rodr\u00edguez, J.F., Casasnovas, J.M., M\u00e9rida, I., and \u00c1vila-Flores, A. (2021). SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells. Cells, 10.","DOI":"10.3390\/cells10123279"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"151682","DOI":"10.1016\/j.anndiagpath.2020.151682","article-title":"Endothelial Cell Damage Is the Central Part of COVID-19 and a Mouse Model Induced by Injection of the S1 Subunit of the Spike Protein","volume":"51","author":"Nuovo","year":"2020","journal-title":"Ann. Diagn. Pathol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5496","DOI":"10.1038\/s41598-022-09410-7","article-title":"SARS-CoV-2 Spike Protein Induces Cognitive Deficit and Anxiety-like Behavior in Mouse via Non-Cell Autonomous Hippocampal Neuronal Death","volume":"12","author":"Oh","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e70023","DOI":"10.1096\/fba.2025-00005","article-title":"Role of NRP1\/HDAC4\/CREB\/RIPK1 Axis in SARS-CoV2 S1 Spike Subunit-Induced Neuronal Toxicity","volume":"7","author":"Sanguigno","year":"2025","journal-title":"FASEB Bioadv."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Kim, E.S., Jeon, M.T., Kim, K.S., Lee, S., Kim, S., and Kim, D.G. (2021). Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells. Viruses, 13.","DOI":"10.3390\/v13102021"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"106700","DOI":"10.1016\/j.cct.2022.106700","article-title":"COVID-19 Vaccines: Considering Sex Differences in Efficacy and Safety","volume":"115","author":"Jensen","year":"2022","journal-title":"Contemp. Clin. Trials"}],"container-title":["Medicina"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1648-9144\/62\/1\/198\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T09:10:10Z","timestamp":1768900210000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1648-9144\/62\/1\/198"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,17]]},"references-count":40,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["medicina62010198"],"URL":"https:\/\/doi.org\/10.3390\/medicina62010198","relation":{},"ISSN":["1648-9144"],"issn-type":[{"value":"1648-9144","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,17]]}}}