Ischemic stroke (IS) is contemplated as a condition that causes a decrease in oxygen and nutrients reaching the brain due to inadequate blood supply to cerebral tissues (Rink and Khanna, 2011). It accounts for about 87 % of all strokes, indicating its dominance in the overall stroke burden (Saini et al., 2021). Ischemic reperfusion (I/R) injury is a complex phenomenon that occurs when blood flow is restored to ischemic tissue after a period of hypoxia, exacerbating damage beyond the initial ischemic insult (Zicola et al., 2021). I/R injuries involve a variety of pathological processes, including inflammatory response, damage to cells (apoptosis and necrosis), oxidative stress, extracellular matrix (ECM) remodeling, and angiogenesis (Zhang et al., 2024). Furthermore, reoxygenation of tissue leads to blood-brain barrier (BBB) disruption, microglial activation, and vasogenic edema (Witt et al., 2008; Zhao et al., 2017). The BBB disruption results in the release of pro-inflammatory cytokines, which then trigger a series of pathological cascades that directly or indirectly cause apoptosis.

Activation of reactive oxygen species induced by reperfusion injury has been recognized as a key contributor to pro-inflammatory responses. Moreover, the activation and polarization of microglia, particularly M1 or M2 phenotypes, have been identified as significant factors in the pathogenesis and recovery from IS (Jiang et al., 2020; Jia et al., 2022). It has been reported that cell death mechanisms are activated within 24h of I/R injury in the brain (Chen et al., 2011; Zhang et al., 2022). There is an imbalance between cell death proteins and cell survival proteins expression post I/R injury (Uzdensky, 2019). CREB (cyclic AMP response element binding protein), a transcription factor associated with antiapoptotic properties, is expressed in the first hour and lasts longer after an ischemic event (Meller et al., 2005). On the other hand, caspases, especially caspase-3, a member of the cysteine protease family—have been identified as key markers of apoptosis. Studies have shown that cytosolic activation of caspase-3 occurs at 20min post-ischemia, and activity remains in the nucleus at 18–24h post-insult (Tenneti and Lipton, 2000). The imbalance between CREB and caspase-3 leads to ischemic cell death (Gerace et al., 2012; Fujii et al., 2014; Huang et al., 2015).

Recently, it has been proven that iron may be a risk factor in the development of cerebral I/R injury (Wang et al., 2021). The presence of free iron intracellularly is highly pro-oxidant in nature, and catalyse the production of hydroxyl radicals, contributing to oxidative stress and cellular damage. Furthermore, iron catalyses the cascade of lipid peroxidation that drives ferroptosis, a recently discovered mechanism of degenerative cell death (Dixon et al., 2012). Clinically, high plasma ferritin, a reliable indicator of tissue iron, and high cerebrospinal fluid (CSF) ferritin have been related to poor outcomes in stroke patients, which indicates the strong relevance of iron in IS (Pankaj et al., 2015). However, the influence of cerebral I/R-mediated iron overload on prime pathological mechanisms involved in early hours of ischemic neuronal death remain unclear.

Iron chelation therapy prevents iron accumulation, restricting the generation of reactive oxygen species (Dixon and Stockwell, 2014). Deferoxamine (DFX) is a high-affinity iron chelator, has demonstrated neuroprotective effects in experimental stroke models (Freret et al., 2006; Gu et al., 2009; Xing et al., 2009; He et al., 2016). The mechanism of action of DFX involves binding and chelating free iron, hemosiderin, and ferritin, which can contribute to its neuroprotective effects (Janjua et al., 2021). Additionally, DFX has been found to scavenge peroxynitrite, protecting against reoxygenation injury in various systems (Sugamura and Keaney, 2011). It has been shown to reduce brain edema, neuronal death/brain atrophy, and neurological deficits induced by intracerebral hemorrhage (ICH; Li et al., 2022). In addition, DFX reduces iron-mediated oxidative stress, neurotoxicity, and secondary brain injury (Xing et al., 2009). Ward et al. demonstrated that DFX can cross the BBB and decrease iron content in both the cerebellum and cerebral cortex in rats (Ward et al., 1995). Interestingly, shreds of evidence suggest that iron participates in ischemia-induced BBB disruption (DeGregorio-Rocasolano et al., 2019; Song et al., 2020) and that DFX can reduce such disruption and bind to iron. Moreover, recent data also suggest that DFX stabilizes the disrupted BBB in electrode implant-induced brain injury (Bennett et al., 2019). Additionally, DFX exhibits anti-inflammatory effects, modulating the immune response and reducing inflammatory effects in spinal cord injury and septic shock, and other inflammatory diseases by modulating cellular chelatable iron (Paterniti et al., 2010; Wang et al., 2015). Also, DFX has shown neuroprotective properties in rat models of subarachnoid and intracerebral hemorrhage (Hishikawa et al., 2008; Wan et al., 2023) and traumatic brain injury (Long et al., 1996). However, the underlying neuroprotective mechanisms of DFX remain unclear. DFX was under clinical trials for ICH [(ClinicalTrials.gov Identifier: NCT02175225); Selim et al., 2019], where it showed positive clinical outcomes along with some minor side effects. Overall, such clinical and preclinical evidence suggests that DFX may be a promising therapy for IS. Hence, the present study aims to investigate whether DFX also affects different pathological events that participate in IS, such as BBB disruption, inflammation, and apoptosis, in early hours of therapeutic window.