This systematic review included English-language studies that evaluated the impact of calcium (Ca²⁺) modulation, for example, calcium channel blockers, calcium chelators, or intracellular regulators of calcium, on long-term outcome in animal models of ischemic stroke. Qualifying studies reported at least one recovery-associated or neuroprotective outcome (e.g., neurological function, infarct size, cognitive or motor recovery, or neuronal survival) with a follow-up duration of at least 30 days.

A systematic review was conducted in PubMed, Scopus, Web of Science, and the Cochrane Library with the pre-specified Medical Subject Headings (MeSH) terms and keywords. Editorials, conference abstracts, meta-analyses, and reviews, as well as studies that failed to report discrete outcome data regarding neuroprotection, were excluded. Studies failing to isolate the effects of modulation of calcium from other interventions administered concomitantly were also excluded.

A comprehensive literature search was conducted across PubMed, Scopus, Web of Science, and the Cochrane Library, covering all records up to June 2025. The search strategy utilized MeSH terms and relevant keywords, including “calcium modulation”, “calcium channel blockers”, “ischemic stroke”, “neuroprotection”, and “functional recovery”. Boolean operators (AND, OR) were used to maximize sensitivity and specificity. Filters were applied to include only peer-reviewed studies published in English.

Two independent reviewers screened all titles and abstracts to identify potentially eligible studies. Full texts of selected articles were then assessed against the inclusion and exclusion criteria. Discrepancies were resolved through discussion or consultation with a third reviewer.

The systematic review was conducted in accordance with the PRISMA guidelines. A comprehensive literature search was performed across the following databases: PubMed, Scopus, Web of Science, and the Cochrane Library (up to June 2025).

Studies were selected based on predefined eligibility criteria, which included animal models of ischemic stroke with at least 30 days of follow-up. The data from the selected studies were extracted using a standardized form, which included study design, intervention details, outcome measures, and key findings.

Risk of bias was assessed using the Risk of Bias in Non-randomized Studies of Interventions (ROBINS-I), which evaluates seven domains:

1.

Bias due to confounding.

Each domain was judged as Low, Moderate, Serious, Critical, or No information risk of bias.

Interventions:

The systematic review included studies using the following interventions:

1.

Calcium-modulating agents, including:

a.

Traditional calcium channel blockers (e.g., L-type blockers).

These interventions were tested in various animal models of ischemic stroke to assess their long-term neuroprotective effects.

A total of 3,120 records were identified through database searching: PubMed (n = 1,230), Scopus (n = 980), Web of Science (n = 720), and the Cochrane Library (n = 190). After removing 820 duplicates and 100 irrelevant publication types (e.g., non-English articles, reviews, or editorials), 2,200 records remained for title and abstract screening. Of these, 2,100 records were excluded due to irrelevance.

The full texts of 100 articles were retrieved and assessed for eligibility. After detailed review, 91 studies were excluded due to: irrelevant outcomes (n = 35), wrong study design (n = 28), or other reasons such as incomplete data or absence of a comparator group (n = 28). Ultimately, 9 studies met the inclusion criteria and were included in the final analysis.

Due to the heterogeneity across the included studies, including differences in study designs, animal models, interventions, outcome measures, and follow-up periods, a meta-analysis was not feasible. The differences in study designs and outcome reporting made it difficult to combine the data in a meaningful way. Therefore, a qualitative systematic review approach was used to synthesize the findings from the selected studies.

The full study selection process is summarized in the PRISMA flow diagram (Figure 1).

The 9 included studies were published between 2015 and 2025 and involved both in vivo and in vitro experimental models of ischemic stroke. Most studies employed middle cerebral artery occlusion (MCAO) models in rodents. Calcium-targeted interventions included L-type calcium channel blockers, magnesium sulfate, EGCG, ischemic preconditioning, small interfering RNA (siRNA) targeting stromal interaction molecule 1 (STIM1), and other novel approaches.

Outcomes assessed included infarct volume, neuronal apoptosis, neurobehavioral scores, intracellular calcium regulation, and molecular pathway activity. A summary of key study characteristics and findings is provided in Table 1.

Across all studies, calcium modulation strategies demonstrated consistent neuroprotective effects. These included reduced infarct volume, decreased expression of apoptosis markers (e.g., cleaved caspase-3), improvement in neurological scores, and restoration of intracellular calcium balance.

Key molecular pathways implicated in these effects included phosphoinositide 3-kinase (PI3K)/AKT signaling, caspase-dependent apoptosis, STIM1/ORAI1-mediated store-operated calcium entry (SOCE), and calcium-sensor proteins such as NCKX2 and hippocalcin.

Combination therapies, such as magnesium sulfate with intra-arterial hypothermia or nanomedicine-based delivery, further enhanced neuroprotection in some models.

The methodological quality of the included studies was generally moderate. Risk of bias was assessed using the ROBINS-I. Most studies had low risk in domains such as outcome measurement and classification of interventions. However, some studies showed concerns related to selection bias, lack of randomization details, or potential confounding. Each domain was classified as Low, Moderate, Serious, Critical, or No information risk of bias.

A full summary of the risk of bias evaluation is provided in Table 2.

This systematic review provides robust preclinical evidence that calcium (Ca²⁺) dysregulation plays a pivotal role in the pathophysiology of ischemic stroke. Targeted calcium modulation strategies, including interventions such as ischemic preconditioning, L-type calcium channel blockers, antioxidants (e.g., EGCG, borneol), and magnesium sulfate, have demonstrated significant neuroprotective benefits. These interventions mitigate intracellular calcium overload, oxidative stress, infarct volume, and neuronal apoptosis [10], leading to improved neurobehavioral outcomes. The underlying mechanisms involve key molecular pathways, including PI3K/AKT signaling, caspase-dependent apoptosis, STIM1/ORAI1-mediated SOCE, and calcium-sensor proteins like NCKX2 and hippocalcin [11]. Genetic silencing of key calcium-regulatory pathways, such as STIM1 and NCKX2, has been shown to exacerbate neurological damage, reinforcing their causal role in stroke pathophysiology. Furthermore, combination therapies and novel delivery approaches, including nanomedicine formulations, have demonstrated enhanced therapeutic efficacy in experimental models [1–7].

Our findings align with established literature indicating that calcium overload is a major contributor to secondary neuronal injury following cerebral ischemia [2, 3, 10, 11]. Early clinical studies investigating calcium channel blockers like nimodipine yielded limited success in treating acute ischemic stroke, likely due to non-specific blockade, poor timing of administration, and adverse hemodynamic effects [12]. However, recent preclinical research has demonstrated more selective and effective calcium modulation strategies [4].

Notably, newer studies have incorporated the role of calcium signaling in glial cells and immune responses, including factors such as hypoxia-inducible factor 1-alpha (HIF1A) and pathways of endoplasmic reticulum stress [e.g., GRP78 (78-kDa glucose-regulated protein)], adding mechanistic depth to the understanding of ischemia-induced calcium toxicity [13, 14]. The use of dual-target or combinatory strategies, such as magnesium sulfate with hypothermia or siRNA with pathway inhibitors, reflects current trends in translational neuroprotection research, which increasingly favors multimodal approaches over single-agent therapies [6, 9].

A major strength of this review is its focus on long-term outcomes and mechanistic insights from recent preclinical models. The inclusion of both in vitro and in vivo studies provides a comprehensive view of calciumʼs neurobiological role across different experimental contexts. The use of a rigorous methodology, including standardized bias assessment, enhances the reliability of the findings.

However, several limitations exist. The heterogeneity of stroke models, intervention protocols, and outcome measures complicates direct comparisons across studies. Due to these variations, a meta-analysis was not feasible, as differences in study designs, animal models, interventions, and follow-up periods made it difficult to combine the data in a meaningful way. As a result, a qualitative systematic review approach was used to synthesize the findings.

Moreover, despite focusing on long-term outcomes, many included studies still emphasized acute-phase results without extended behavioral follow-up. The exclusive reliance on preclinical data also limits the generalizability of the findings to human stroke patients. Lastly, potential publication bias and incomplete reporting in some studies cannot be excluded.

The preclinical evidence presented herein suggests that targeting calcium signaling pathways holds promise as an adjunctive approach for ischemic stroke management. Interventions such as calcium channel blockers, magnesium sulfate, and natural antioxidants may complement existing reperfusion therapies by mitigating delayed neuronal damage [7, 12]. However, translating these findings into clinical practice necessitates careful consideration of several factors:

Timing and dosage: Determining the optimal timing and dosage of calcium-targeted interventions is crucial to maximize neuroprotective effects while minimizing potential adverse outcomes.

Future studies should prioritize clinical validation of calcium-targeted therapies, particularly in combination with anti-inflammatory or antioxidant strategies. Longitudinal studies assessing functional outcomes beyond the acute phase are urgently needed. Research should also investigate calcium’s role in glial activation, immune modulation, and synaptic remodeling to capture the full spectrum of post-stroke neurobiology. Lastly, the development of calcium-related biomarkers may enable early diagnosis, patient stratification, and personalized treatment strategies [15–17].

Calcium (Ca²⁺) dysregulation is a central mechanism underlying neuronal injury in ischemic stroke, contributing to excitotoxicity, oxidative stress, and apoptosis. This systematic review underscores consistent preclinical evidence demonstrating that calcium modulation, through agents such as L-type calcium channel blockers, magnesium sulfate, antioxidants (e.g., EGCG, borneol), ischemic preconditioning, and genetic modulation, can confer long-term neuroprotective effects in experimental stroke models.

These interventions appear to reduce infarct volume, preserve neuronal integrity, and improve functional recovery by modulating key molecular pathways, including PI3K/AKT, STIM1/ORAI1-mediated SOCE, caspase-dependent apoptosis, and calcium-sensor proteins like NCKX2 and hippocalcin. Moreover, combination therapies and nanomedicine-based delivery approaches have shown potential to further enhance treatment efficacy, offering promising avenues for improving neuroprotective strategies [14].

Despite these promising findings, clinical translation remains limited due to several factors, including a lack of standardized outcome measures, heterogeneous experimental designs, and a general lack of human data. Moreover, variations in animal models, intervention protocols, outcome measures, and follow-up periods prevented the conduct of a meta-analysis. As a result, we used a qualitative systematic review approach to synthesize the available evidence and present key trends in calcium modulation for ischemic stroke recovery.

Future research should prioritize clinical validation of calcium-targeted therapies, focusing on standardized protocols and the exploration of calcium-based biomarkers [18]. These efforts could guide the development of personalized stroke recovery therapies. Longitudinal human studies, including large-scale clinical trials, are urgently needed to assess the safety, efficacy, and optimal treatment windows for these therapies.

In conclusion, calcium modulation represents a promising strategy for adjunctive neuroprotection in ischemic stroke. As the field transitions towards multimodal and personalized stroke management, calcium-targeted therapies deserve increased attention in both translational and clinical research, offering hope for improving stroke recovery outcomes and patient quality of life.