The majority of approved therapies for many diseases are developed to target their underlying pathophysiology. Understanding disease pathophysiology has thus proven vital to the successful development of clinically useful medications. Stroke is generally accepted as the leading cause of adult disability globally and ischemic stroke accounts for the most common form of the two main stroke types. Despite its health and socioeconomic burden, there is still minimal availability of effective pharmacological therapies for its treatment. In this review, we take an in-depth look at the etiology and pathophysiology of ischemic stroke, including molecular and cellular changes. This is followed by a highlight of drugs, cellular therapies, and complementary medicines that are approved or undergoing clinical trials for the treatment and management of ischemic stroke. We also identify unexplored potential targets in stroke pathogenesis that can be exploited to increase the pool of effective anti-stroke and neuroprotective agents through de novo drug development and drug repurposing.

Promising preclinical stroke research has not yielded meaningful and significant success in clinical trials. This lack of success has prompted the need for refinement of preclinical studies with the intent to optimize the chances of clinical success. Regenerative medicine, especially using mesenchymal stem/stromal cells (MSCs), has gained popularity in the last decade for treating many disorders, including central nervous system (CNS), such as stroke. In addition to less stringent ethical constraints, the ample availability of MSCs also makes them an attractive alternative to totipotent and other pluripotent stem cells. The ability of MSCs to differentiate into neurons and other brain parenchymal and immune cells makes them a promising therapy for stroke. However, these cells also have some drawbacks that, if not addressed, will render MSCs unfit for treating ischaemic stroke. In this review, we highlighted the molecular and cellular changes that occur following an ischaemic stroke (IS) incidence and discussed the physiological properties of MSCs suitable for tackling these changes. We also went further to discuss the major drawbacks of utilizing MSCs in IS and how adequate priming using both hypoxia and interleukin-1 can optimize the beneficial properties of MSCs while eliminating these drawbacks.

Background: Hypoxic–ischaemic 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 < 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-1β, TNF-α, and HIF-1α as key drivers during OGD, with additional regulators emerging during reoxygenation. TNF-α and β-oestradiol were consistently identified across time points, while TGF-β1 and NTRK1 became prominent during peak injury and later phases. Analysis of secreted factors showed increased release of inflammatory (TNF-α) and neurotrophic (β-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–ischaemic 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-α, IL-β, and HIF-1α), context-specific (NTRK1 and TGF-β) and novel (β-oestradiol) regulators of the injury response with potential relevance for therapeutic targeting.

Background and Purpose: Human mesenchymal stem cells (MSCs) show promise for treating ischaemic and inflammatory conditions. However, their clinical utility is limited by issues like poor migration, engraftment, rapid elimination, and slow functional recovery post-thaw. We investigated whether dual priming of human bone marrow-derived MSCs (hBMSCs) with IL-1α and CoCl2 will enhance their therapeutic potential. Experimental Approach: hBMSCs were primed individually or in combination with IL-1α and CoCl2. Their conditioned medium (CM) was then analysed for secreted biomolecules, including G-CSF, IL-8, BDNF, β-NGF, ICAM-1, and TNF-α. Two in vitro models using BV2 cells were employed: one to assess anti-inflammatory efficacy by treating LPS-induced inflammation with CM, and another to evaluate cytoprotective effects by exposing cells to oxygen and glucose deprivation/reperfusion in the presence of CM. Markers of inflammation, neuroinflammation, and cell death were subsequently measured. Key Finding: Priming significantly altered hBMSCs secretion profiles: IL-8 decreased (~3-fold), while β-NGF (1.3-1.9-fold) and ICAM-1 (2.7-4-fold) increased. G-CSF (100-400 pg/ml) and BDNF (~165 pg/ml) became detectable. TNF-α response varied, decreasing with IL-1α priming but increasing with dual priming. In both LPS-induced inflammation and OGD/reperfusion models, CM from primed hBMSCs significantly reduced IL-6, IL-1β, enhanced IL-10, and decreased MMP-9 expression. Notably, while all hBMSCs CMs alleviated LPS-induced cell death, only dual-primed CM demonstrated significant cytoprotective effects in the ischaemia/reperfusion model. Conclusion: Dual priming of hBMSCs with both hypoxia (CoCl2 and IL-1α significantly enhanced their therapeutic potential for managing hypoxic-ischaemic conditions and offers a promising strategy for developing more effective cell-based therapies for Ischaemic stroke

Background and Purpose: Human mesenchymal stem cells (MSCs) show promise for treating ischaemic and inflammatory conditions, yet their clinical utility is limited by a failure to replicate preclinical efficacy. We hypothesized that dual priming of human bone marrow-derived MSCs (hBMSCs) with interleukin-1alpha (IL-1α) and cobalt chloride, CoCl ₂ (a hypoxia mimetic) would potentiate their therapeutic efficacy for hypoxic-ischaemic conditions. Experimental Approach: hBMSCs were primed individually or in combination. Their conditioned media (CM) was analysed for relevant secreted biomolecules. To test the anti-inflammatory and cytoprotective properties, primed-hBMSCs-derived CM was applied to LPS-treated BV2 microglial cells ( in vitro inflammation model) and BV2 cells exposed to oxygen glucose deprivation (OGD) ( in vitro hypoxic injury model). The neuroprotective effect was evaluated by assessing neuronal death (using NLCs) following exposure to OGD/reoxygenation (OGD/R) in the presence of various CMs. Key Finding: Priming significantly altered hBMSCs’ secretion profiles. IL-1α triggered significant ( p<0.05 ) release of inflammatory mediators/cytokines, notably TNF-α, ICAM-1, and G-CSF. Except for G-SCF, dual-priming did not potentiate the release of these inflammatory molecules. However, while single priming with either IL-1α or hypoxia did not induce remarkable secretion of the neurotrophins β-NGF and BDNF, the dual-priming resulted in significant ( p<0.05 ) and additive secretion of these neurotrophins. In the LPS model, CM from dual-primed hBMSCs significantly reduced the secretion of pro-inflammatory cytokines, TNF-α and IL-6. In the hypoxia model, priming did not significantly influence the marked reduction ( p<0.05 ) of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Lastly, CMs from primed hBMSCs significantly (p<0.05) reduced neuronal cytotoxicity, yet only CM from dual-primed hBMSCs significantly (p<0.05) preserved the viability of NLCs following 24 h reperfusion after OGD. Conclusion: Dual priming of hBMSCs with both hypoxia and IL-1α significantly enhanced their therapeutic potential for managing hypoxic-ischemic conditions and offers a promising strategy for developing more effective cell-based therapies for ischaemic stroke.

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.