{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T06:14:46Z","timestamp":1790835286302,"version":"4.1.0"},"reference-count":59,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T00:00:00Z","timestamp":1782345600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathophysiology"],"abstract":"<jats:p>Background: Hypoxic\u2013ischaemic brain injury (HI) is a major contributor to neurological deficits following stroke. Understanding what happens to the smallest functional and structural unit of the central nervous system in the face of oxygen and nutrient deprivation is essential to fully comprehend the pathogenesis of diseases and disorders associated with HI, such as ischaemic stroke. Aim: The aim of this study was to develop a robust in vitro tool for initial screening of potential therapeutics and identification of diagnostic markers of brain hypoxic injury. Methods: This study details and validates a comprehensive protocol for modelling HI using differentiated SH-SY5Y neuroblastoma cells (Neuron-like Cells, NLCs). First, we optimized the differentiation process and confirmed the maturity and purity of NLCs via standard molecular markers. The NLCs exhibited functional excitotoxicity, demonstrating a graded cell death response to N-methyl-D-aspartate (NMDA), thus validating their functional application. To simulate HI, we initially optimized the oxygen-glucose deprivation (OGD) treatment using graded concentrations of CoCl2 (0.125 mM to 2 mM) in glucose-free media. The validated NLCs were then subjected to the refined OGD protocol (1 mM CoCl2 in glucose-free media) for 3 h, followed by various periods of reoxygenation (1 h, 3 h, 6 h, 12 h, 18 h, and 24 h). Result: Bulk RNA-sequencing revealed a distinct temporal transcriptional response to HI. Injury-associated genes, including heat shock proteins and stress markers, were significantly (p &lt; 0.05) upregulated at 3 h of reoxygenation, peaked at 6 h, and declined thereafter, remaining above baseline at 24 h. Upstream regulator analysis identified IL-1\u03b2, TNF-\u03b1, and HIF-1\u03b1 as key drivers during OGD, with additional regulators emerging during reoxygenation. TNF-\u03b1 and \u03b2-oestradiol were consistently identified across time points, while TGF-\u03b21 and NTRK1 became prominent during peak injury and later phases. Analysis of secreted factors showed increased release of inflammatory (TNF-\u03b1) and neurotrophic (\u03b2-NGF, BDNF, VEGF) mediators with reoxygenation, while maximal cell death occurred at 24 h. Conclusions: This study identifies a transient, time-dependent transcriptional cascade following hypoxic\u2013ischaemic injury, highlighting a critical window for early neuronal response. The model provides a reproducible platform for studying neuronal injury and recovery, and identifies known (TNF-\u03b1, IL-\u03b2, and HIF-1\u03b1), context-specific (NTRK1 and TGF-\u03b2) and novel (\u03b2-oestradiol) regulators of the injury response with potential relevance for therapeutic targeting.<\/jats:p>","DOI":"10.3390\/pathophysiology33030043","type":"journal-article","created":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T00:49:23Z","timestamp":1782434963000},"page":"43","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Differentiated SH-SY5Y Model of Hypoxic\u2013Ischaemic Injury Reveals Dynamic Transcriptomic Responses During Reoxygenation"],"prefix":"10.3390","volume":"33","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4066-7937","authenticated-orcid":false,"given":"Maryam Adenike","family":"Salaudeen","sequence":"first","affiliation":[{"name":"Division of Neuroscience, Faculty of Biology, Medicine, and Health, School of Biological Sciences, University of Manchester, Manchester M13 9PL, UK"},{"name":"Geoffrey Jefferson Brain Research Centre, Manchester Academic Health Science Centre, Northern Care Alliance NHS Foundation Trust, University of Manchester, Manchester M13 9PL, UK"},{"name":"Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria 810211, Nigeria"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9646-4456","authenticated-orcid":false,"given":"Stuart M.","family":"Allan","sequence":"additional","affiliation":[{"name":"Division of Neuroscience, Faculty of Biology, Medicine, and Health, School of Biological Sciences, University of Manchester, Manchester M13 9PL, UK"},{"name":"Geoffrey Jefferson Brain Research Centre, Manchester Academic Health Science Centre, Northern Care Alliance NHS Foundation Trust, University of Manchester, Manchester M13 9PL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emmanuel","family":"Pinteaux","sequence":"additional","affiliation":[{"name":"Division of Neuroscience, Faculty of Biology, Medicine, and Health, School of Biological Sciences, University of Manchester, Manchester M13 9PL, UK"},{"name":"Geoffrey Jefferson Brain Research Centre, Manchester Academic Health Science Centre, Northern Care Alliance NHS Foundation Trust, University of Manchester, Manchester M13 9PL, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1038\/nrm2236","article-title":"The third dimension bridges the gap between cell culture and live tissue","volume":"8","author":"Pampaloni","year":"2007","journal-title":"Nat. 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