Ischemic stroke is a leading cause of disability and death worldwide, with the cerebellum being particularly vulnerable due to its critical role in motor coordination and balance [5]. Neuronal damage in cerebellar stroke arises from oxygen-glucose deprivation (OGD), which triggers complex pathological mechanisms such as ionic dysregulation, altered metabolism, oxidative stress, and inflammation [21, 22]. Purkinje cells, essential for cerebellar function, are highly susceptible to hypoxic injury, leading to severe and often irreversible consequences for stroke survivors [5, 23]. Current stroke therapies, such as thrombolysis or thrombectomy, primarily focus on restoring blood flow and preventing recurrence [24], with few options that directly target the molecular cascades underlying neuronal injury. Thus, developing cell protective strategies that mitigate cell death could significantly improve patient outcomes.

In this study, we investigated the potential cell protective effects of Edaravone, a radical scavenger known for its efficacy in mitigating oxidative stress, particularly in the context of stroke [10, 25, 26]. Using an OGD model in organotypic slice cultures from rat cerebellum and hippocampus, we simulated ischemic stroke conditions and evaluated Edaravone’s ability to reduce cellular damage [19]. The hippocampus, a region highly sensitive to hypoxia [27], was used as a comparative structure in this study, as prior studies have demonstrated Edaravone’s neuroprotective effects specifically in hippocampal tissue [28,29,30]. This provided a robust benchmark to assess the strength of Edaravone’s protective effects in the cerebellum.

Our findings confirm that OGD effectively induces hypoxia, as evidenced by significant upregulation of HIF-1α and HIF-2α mRNA in both cerebellar and hippocampal tissues [31]. These markers are crucial indicators of hypoxic response [32]. Interestingly, while both tissues exhibited elevated HIF mRNA levels, the protein analysis revealed tissue-specific differences. In the cerebellum, HIF-2α protein levels were significantly increased, whereas HIF-1α was significantly upregulated in the hippocampus. These discrepancies could be attributed to the cellular composition of the tissues. HIF-1α is predominantly expressed in neurons, whereas HIF-2α is primarily found in non-neuronal cells such as endothelial cells and astrocytes [33]. Especially endothelial cells play a central role in the expression of HIF-2α under hypoxic conditions [34]. Given the cerebellum’s relatively higher density of endothelial cells, the stronger HIF-2α response likely reflects the endothelial contribution to hypoxic adaptation in this tissue [35]. The lack of significant HIF-1α upregulation in the cerebellum at the 4-hour time point might be explained by differences in mechanisms of proteostasis or the metabolic activity of cerebellar neurons compared to hippocampal neurons. This difference could be attributed to the fact that the hippocampus is a brain region that shows a particularly strong accumulation of HIF-1α under hypoxia [36]. Additionally, the higher LDH levels observed in cerebellar cultures after OGD suggest more pronounced cellular damage compared to the hippocampus, which could significantly influence the observed differences in HIF expression. The selective loss of cerebellar granule cells, as demonstrated in our previous work [19], may have led to a reduced contribution of HIF-1α-producing neuronal populations, whereas the remaining non-neuronal cells, such as astrocytes and endothelial cells, could continue to upregulate HIF-2α. The greater extent of cell damage in the cerebellum could thus contribute to the differential expression patterns observed. While cerebellar neurons might degrade HIF-1α protein more rapidly during reoxygenation, limiting its accumulation, the cerebellum’s hypoxic response might be dominated by endothelial pathways, reducing the relative contribution of neuronal HIF-1α. These tissue-specific dynamics highlight the complexity of the cerebellum’s response to hypoxia and suggest that its vulnerability might not solely depend on neuronal factors. Further studies focusing on cell-type-specific dynamics of HIF expression and stability are needed to clarify these mechanisms and their implications for therapeutic interventions like Edaravone.

The selection of the 4 h OGD time point for Edaravone treatment was based on our observation that hypoxia-induced cell responses were well-established at this stage. Although earlier cellular responses were detectable after 1 h, longer OGD durations likely provide a more clinically relevant scenario, as stroke patients typically present after a delay [37, 38]. This approach aligns with therapeutic windows for lysis or thrombectomy, making Edaravone a potential adjunct treatment during revascularization and reoxygenation [24]. Future studies should explore the timing and efficacy of Edaravone administration, including pre-hypoxia applications, particularly in high-risk stroke models or surgical scenarios where ischemic events are anticipated. Considering its potent antioxidant properties and established safety profile, Edaravone has the potential to serve as a prophylactic agent, preconditioning neuronal tissue against ischemic injury, mitigating oxidative damage, and reducing the impact of anticipated hypoxic conditions [39].

While previous studies have already indicated the neuroprotective effect of Edaravone in hypoxic cell damage [40], in our study Edaravone also demonstrated significant protective effects in both cerebellar and hippocampal tissues, as indicated by reduced LDH levels in OGD-treated samples. LDH release, a marker of cellular damage, is triggered by membrane permeability loss during hypoxia [41]. Edaravone significantly reduced LDH levels in both tissues, indicating comparable cellular protection in cerebellar and hippocampal tissues, a finding consistent with previous studies on hippocampal protection [28, 30]. However, we observed that the cerebellum experienced a higher percentage increase in LDH levels compared to the hippocampus under OGD conditions. This could suggest greater vulnerability to hypoxia in the cerebellum, though the hippocampus is known to be highly sensitive to hypoxic damage [42]. While the cerebellum showed more pronounced cellular damage, the hippocampus may suffer more from functional impairments due to its involvement in cognitive functions such as memory and learning [43]. This heightened susceptibility is attributed to the vulnerability of hippocampal pyramidal cells and fast-spiking interneurons, which are essential for hippocampal function [44, 45]. Their loss leads to significant functional deficits [43]. Since our assay focused on cellular damage (LDH release), rather than functional deficits, we may not fully capture the hippocampal vulnerability. Future studies incorporating functional assessments would provide a clearer picture of Edaravone’s protective effects.

In addition to its effects on cellular damage, Edaravone significantly reduced ROS levels in both cerebellar and hippocampal slice cultures, emphasizing its role as an effective radical scavenger [10]. OGD conditions induced an increase in ROS production, which persisted through the reoxygenation phase, further exacerbating cell damage. Edaravone’s ability to mitigate this oxidative burst highlights its therapeutic potential in limiting reperfusion injury, a major contributor to neuronal death following stroke [9, 46]. Notably, cerebellar cultures showed higher ROS levels than hippocampal cultures after 4 h OGD, which might reflect the cerebellum’s distinct cellular composition and metabolic demands. Previous studies have shown that the cerebellum, along with the brain stem, exhibits the highest basal rate of ROS production [47]. The relatively high density of astrocytes in the hippocampus, which are known for their antioxidative metabolism and increased glutathione synthesis, may contribute to lower ROS levels under hypoxic conditions [48]. In contrast, Purkinje cells, with their extensive dendritic arborization and elevated metabolic activity, may further amplify ROS production in the cerebellum [49]. These factors could explain the cerebellum’s heightened oxidative response and its relatively higher ROS levels during hypoxia. Our findings indicate that Edaravone’s radical-scavenging effects were more pronounced in the cerebellum, potentially due to the higher initial ROS levels, which provide a greater window for antioxidative intervention. However, excessive ROS levels might also overwhelm Edaravone’s scavenging capacity, partially contributing to less pronounced reduction in LDH levels observed in the cerebellum. It is important to note that elevated ROS levels do not necessarily correlate with greater functional damage. Recent studies have shown that following OGD in hippocampal slice cultures, Edaravone treatment did not attenuate the decline in neuronal transmission [50]. Since our study focused on quantitative cell survival rather than functional assessment, this represents a limitation of our approach and should be further investigated in future studies. The cited study also highlights the importance of superoxide radicals and suggests that Edaravone, as a non-specific radical scavenger, may be less effective compared to other agents specifically targeting super oxides. This aspect requires further exploration to determine the most effective antioxidant strategy.

Mitochondrial dysfunction plays a central role in ischemic neuronal damage, as OGD disrupts oxidative phosphorylation and ATP production [51]. Our findings reveal tissue-specific differences in mitochondrial response to OGD and Edaravone treatment. In cerebellar tissue, Edaravone primarily preserved tissue integrity during reperfusion, as indicated by higher OCR levels after normalization, suggesting that the observed reductions in raw OCR values after OGD were primarily due to cell loss rather than mitochondrial dysfunction. The higher LDH release in cerebellar tissues under OGD conditions reflects greater cellular damage, consistent with the heightened vulnerability of metabolically active Purkinje cells to hypoxic injury [52], which might explain why Edaravone did not influence mitochondrial respiration in cerebellar tissue. In contrast, hippocampal cultures exhibited a distinct mitochondrial response. Both, raw and DNA-normalized OCR data showed a significant enhancement of mitochondrial function in the OGD + Edaravone group which resulted in increased basal respiration and glucose-induced maximal respiration. This suggests that Edaravone not only maintains cellular integrity but also actively enhances mitochondrial activity in this tissue. This may involve mechanisms such as increased metabolic efficiency or mitochondrial biogenesis, potentially mediated by transcription factors like PGC-1α, which regulate mitochondrial adaptation to oxidative stress [53]. The improved mitochondrial function in the hippocampus is further supported by reduced LDH release and lower ROS levels, suggesting a more robust recovery of metabolic and antioxidative capacity. Our findings further support a tissue-specific role of Edaravone in stroke recovery. In cerebellar tissue, the lack of significant changes in DNA-normalized OCR across all conditions suggests that Edaravone’s primary function is cell preservation rather than direct mitochondrial enhancement. The recovery of basal OCR in raw data, despite unchanged DNA-normalized OCR, reinforces this notion, indicating that Edaravone prevents cell loss but does not necessarily improve mitochondrial respiration per cell. In contrast, hippocampal slices exhibited a clear enhancement of mitochondrial function, particularly in glucose-induced OCR, suggesting Edaravone promotes metabolic efficiency post-ischemia. The fact that pyruvate-driven respiration remained unaffected suggests that Edaravone’s effect is primarily linked to glucose metabolism, which represents the primary source of energy for the brain as it fuels the TCA cycle to enhance oxidative phosphorylation. These findings highlight the importance of region-specific metabolic responses in ischemic brain injury and recovery. Edaravone’s effect in the cerebellum appears more structural, whereas in the hippocampus, it enhances cellular metabolism and mitochondrial function, which could have implications for functional recovery and neuroplasticity. These insights emphasize the need for tailored therapeutic approaches depending on the affected brain region, particularly in stroke therapy where metabolic and cellular vulnerabilities differ. The interplay between ROS scavenging and mitochondrial function is particularly evident in the hippocampus. The marked improvement in mitochondrial respiration in hippocampal slices treated with Edaravone suggests that ROS reduction facilitated mitochondrial recovery and may have activated pathways supporting mitochondrial repair or biogenesis. In contrast, the cerebellum’s less efficient mitochondrial recovery, as indicated by basal respiration as well as glucose-induced and pyruvate-induced maximal respiration, could reflect a diminished capacity for adaptive responses under hypoxic and/or OGD stress, potentially due to its distinct cellular composition and metabolic profile [49].

These findings suggest potential tissue-specific roles for Edaravone in stroke therapy. In the cerebellum, Edaravone may primarily help mitigate cell loss and reduce post-stroke atrophy, which could contribute to preserving motor coordination. In the hippocampus, the observed enhancement of mitochondrial function raises the possibility that Edaravone supports cognitive recovery and neuroplasticity. This highlights the broader potential of Edaravone’s neuroprotective effects, which might extend beyond ROS scavenging to include a role in preventing mitochondrial dysfunction. The observed regional disparities further emphasize the importance of tailoring therapeutic strategies to the specific vulnerabilities and recovery capacities of different brain regions. During reperfusion, where oxidative stress and mitochondrial impairment contribute significantly to secondary injury, Edaravone’s ability to mitigate ROS and support mitochondrial recovery highlights its potential as a region-specific therapeutic agent.

Furthermore Edaravone’s neuroprotective effects go beyond ROS scavenging, potentially involving the prevention of brain oedema and modulation of inflammatory pathways [54, 55]. These additional mechanisms could be particularly relevant in cerebellar stroke, where oedema and brainstem compression pose significant risks [56]. While this study focused on cellular damage, future in vivo studies should investigate Edaravone’s broader therapeutic potential, particularly its role in preventing oedema and managing inflammation.

Edaravone might also have potential applications beyond ischemic stroke. For instance, during myocardial infarction or cardiac pump failure, reduced cardiac output leads to cerebral hypoxia, which our OGD model simulates [57]. Edaravone could offer neuroprotection in these contexts, particularly during resuscitation when reperfusion injury may exacerbate neuronal damage. Additionally, its application in perinatal and neonatal asphyxia, where therapeutic hypothermia remains the only treatment for reducing long-term neurological damage, could represent a critical area for future investigation [58].

In summary, our findings indicate that Edaravone provides significant cell protection in both cerebellar and hippocampal tissues under OGD conditions, primarily through its antioxidant properties. These results suggest that Edaravone holds promise as a therapeutic agent for cerebellar stroke, particularly in combination with reperfusion strategies. Future studies should explore Edaravone’s full therapeutic potential in vivo, including its effects on functional recovery, oedema prevention, and broader clinical applications in hypoxic conditions beyond stroke.