Modelling Hypoxic‐Ischaemic Injury in Differentiated SH‐SY5Y Cells: Integrated Transcriptomic and Functional Analysis Reveals a Transient Transcriptional Peak at 6 Hours of Reoxygenation

Background: Hypoxic-ischaemic brain injury, (HIBI), 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 that are associated with HIBI, as well as serve as a tool for initial screening of potential therapeutics and identification of diagnostic markers. Aim: The aim of this study was to develop a robust in vitro model for mechanistic investigation of the effect of HIBI on neurons. Method: This study details and validates a comprehensive protocol for modelling HIBI 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), validating their functional utility. To simulate HIBI, we initially optimized the oxygen-glucose deprivation (OGD) component using graded concentrations of CoCl2 (0.125mM to 2mM) in glucose-free media. The validated NLCs were then subjected to the refined OGD protocol (1mM CoCl₂ in glucose-free media) for 3 hours, followed by various periods of reoxygenation (1h, 3h, 6h, 12h, 18h, and 24h). Result: Bulk RNA-sequencing analysis revealed a critical temporal pattern in the transcriptional response to injury. Specifically, majority of the injury-related gene expression, including heat shock proteins, stress markers, and cell death were significantly (p<0.05) upgraded at the third hour of reoxygenation, peaked dramatically at the 6th hour, and then rapidly subsided to levels often higher than the baseline at 24 hours. Surprisingly, RNAseq revealed the emergence of transforming growth factor-beta 1 (TGF-β1), a known regulator of glia-scarring, as an upstream regulator after 6 h of reoxygenation. Analysis of the supernatant revealed a corresponding translational pattern, where cell death was most significant after 24 h reoxygenation, whereas the secretion inflammatory cytokine TNF-α, and neuroprotective biomolecules, β-NGF, BDNF, and VEGF increased as reoxygenation time increased. Conclusion: This study reveals the existence of a narrow transient transcriptional cascade, highlighting a critical window for initial damage and cellular response. It also establishes a highly reproducible NLC-based HIBI model and provides a comprehensive transcriptomic blueprint of the immediate and sub-acute neuronal response, pointing toward known and novel upstream regulators of the injury cascade for future therapeutic targeting.