Probiotics and Seizure Susceptibility: A Systematic Review and Meta-Analysis of Preclinical Epilepsy Models

Leila Simani 1

Etrat Hooshmandi 2✉ Phone+98-713-6281572 Emailehoshmandi@gmail.com

Razieh Hajisoltani 3✉ Phone+98-21-86704589 Emailrazieh.hajisoltani@gmail.com

Sedighe Hooshmandi 4

1 Department of Neurology New York University Grossman School of Medicine New York NY USA

Clinical Neurology Research Center Shiraz University of Medical Sciences P.O. Box: 7193635899 Shiraz Iran

Physiology Research Center Iran University of Medical Sciences Tehran Iran

Medical Imaging Research Center, Department of Radiology Shiraz University of Medical Sciences Shiraz Iran

Leila Simani ¹, Etrat Hooshmandi ^2*, Razieh Hajisoltani ^3*, Sedighe Hooshmandi⁴

¹ Department of Neurology, New York University Grossman School of Medicine, New York, NY, USA.

² Clinical Neurology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

³ Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran.

⁴ Medical Imaging Research Center, Department of Radiology, Shiraz University of Medical Sciences, Shiraz, Iran.

*Corresponding authors:

1. Etrat Hooshmandi

Clinical Neurology Research Center, Shiraz University of Medical Sciences,

P.O. Box: 7193635899, Shiraz, Iran.

Tel/Fax: +98-713-6281572

E-mail address: ehoshmandi@gmail.com

2. Razieh Hajisoltani

Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran.

Tel/Fax: +98-21-86704589

E-mail address: razieh.hajisoltani@gmail.com

Abstract

Background

The gut microbiota can affect neuronal excitability, inflammation, and oxidative balance via the gut-brain axis, shaping seizure susceptibility. To translate these mechanistic findings into practical clinical approaches, we need a synthesis of preclinical evidence on microbiome-based interventions.

Objective

A systematic review and meta-analysis to examine probiotics' putative anticonvulsant, anti-inflammatory, antioxidant, and neuroprotective properties in rodent models.

Methods

An extensive, systematic search of online databases was conducted up to July 2025 to identify eligible animal studies in which probiotics were administered in seizure models. Reported outcomes included seizure latency, duration, severity, and frequency, as well as inflammation, oxidative stress, and behavioral measures. Where necessary, outcome data were standardized across studies before pooling.

Results

Of the 24 studies that met the inclusion criteria, 19 provided sufficient data to be included in the meta-analysis. Probiotics significantly increased seizure latency (MD = 22.09; 95%CI: 10.52 to 33.67), and reduced seizure severity (MD= -1.08; 95%CI: −1.39 to − 0.76) and duration (MD= -23.19; 95%CI: −35.56 to − 10.82). Probiotics significantly reduced IL-1β, IL-6, and TNF-α levels while MDA showed a non-significant trend toward reduction (p = .076). Behaviorally, improvements in spatial learning (p < 0.05) and reduced anxiety-like behavior (p < 0.001) were observed.

Conclusion

Probiotic supplementation appears to exert anticonvulsant, anti-inflammatory, antioxidant, and behavioral benefits in preclinical epilepsy models, although the evidence is heterogeneous and limited to animal studies. Mechanistic evidence indicates modulation of the gut–brain axis, enhanced GABAergic signaling, and improved mitochondrial function. These findings support further investigation of specific probiotic formulations as promising adjunct candidates in well-designed, mechanism-driven clinical trials.

Keywords:

Probiotics

Seizure

Epilepsy

Inflammation

Oxidative Stress

Cognition

1. Introduction

The gut microbiota plays a crucial role in the pathophysiology of epilepsy by influencing inflammatory, neurochemical, and barrier mechanisms within the central nervous system (Arulsamy et al. 2020; Li et al. 2024; Zhu et al. 2024). This bidirectional gut-brain axis communication network involves multiple pathways, including the vagus nerve, immune system, neuroendocrine signaling, and microbial metabolites (Li et al. 2024). The gut microbiome, comprising trillions of microorganisms residing in the intestinal tract, modulates brain function by producing neurotransmitters, short-chain fatty acids (SCFAs), and inflammatory mediators that can directly influence neuronal excitability and seizure susceptibility (Kundu et al. 2023).

Clinical studies indicate that epilepsy is associated with gut microbiome dysbiosis, characterized by reduced microbial diversity, depletion of beneficial taxa (e.g., Bifidobacterium and Lactobacillus), and enrichment of pro-inflammatory bacteria (Arulsamy et al. 2020; Riva et al. 2025). These alterations are more pronounced in drug-resistant epilepsy, correlate with seizure severity (Riva et al. 2025), and may contribute to ketogenic diet–mediated antiepileptic effects (Dahlin et al. 2024; Li et al. 2024).

Preclinical studies in rodent models have provided encouraging evidence for the anticonvulsant and neuroprotective effects of various probiotic strains in status epilepticus models. These studies, using chemically induced SE with agents such as pilocarpine, kainic acid (KA), or pentylenetetrazol (PTZ), have demonstrated that probiotic supplementation can reduce seizure severity, decrease neuronal damage, and improve behavioral outcomes (Zhu et al. 2024). Proposed mechanisms include modulation of microglial activation, reduction of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), enhancement of antioxidant defenses, and promotion of neuroplasticity through the expression of neurotrophic factors. Gut microbiota metabolites influence seizure thresholds through the modulation of neurotransmitter systems (particularly GABAergic (gamma Aminobutyric acid) signaling), maintenance of blood-brain barrier integrity, regulation of immune signaling, and neuroprotective lipid production (Zhang et al. 2022; Yang et al. 2024).

Despite these promising findings, several critical knowledge gaps limit the translation of preclinical evidence to clinical practice. The existing preclinical literature presents heterogeneous findings regarding the efficacy of different probiotic strains, administration protocols (including timing relative to seizure onset, treatment duration, and dosing), and outcome measures, making it challenging to identify optimal therapeutic strategies. The mechanisms underlying probiotic-mediated neuroprotection in epileptic seizure remain incompletely understood, and the relative contributions of different bacterial strains and their metabolites to therapeutic effects have not been systematically characterized. Furthermore, no previous systematic review has specifically examined the effects of probiotic supplementation on seizure activity and neuroprotection in rodent models of epilepsy, representing a significant gap in evidence synthesis that limits informed clinical trial design and therapeutic development.

Therefore, the aim of this systematic review and meta-analysis was threefold: (1) to quantify the effects of probiotic administration on seizure outcomes in rodent models of epilepsy as primary endpoints; (2) to evaluate secondary outcomes including inflammatory cytokines, oxidative stress and antioxidant markers, and behavioral/cognitive measures; and (3) to summarize the main mechanistic pathways by which probiotics may modulate the gut–brain axis and influence seizure susceptibility. A rigorous synthesis of preclinical evidence is essential before launching large clinical trials, as it clarifies mechanisms, helps prioritize specific strains and dosing strategies, and can reduce unnecessary animal and human experimentation.

2. Methods

This systematic review and meta-analysis protocol were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.

2.1. Search Strategy

A comprehensive literature search was conducted across multiple electronic databases, including PubMed/MEDLINE, Web of Science, Scopus, Embase, and ProQuest. The search encompassed all studies published in the database from database inception through July 2025 and was limited to English-language articles.

The search combined terms related to seizures (e.g., "Seizure", "Epilepsy"), probiotics (e.g., "Probiotics", "Probiotic agent"), and animal models (e.g., "Animals", "Rodent", "Rats", "Mice"). Boolean operators, controlled vocabulary (MeSH/Emtree terms), and field tags were used. The full search strings for each database are provided in Online Resource as Supplementary Appendix 1. Reference lists of included studies and relevant review articles were manually screened to identify additional eligible studies. Grey literature was searched through conference abstracts, dissertation databases, and research registries to minimize publication bias.

2.2. Eligibility Criteria

Studies were included if they were original research articles reporting experimental studies in rodent models in which rodent species (rats or mice) of any strain, age, or sex were subjected to experimentally induced seizures. Eligible studies investigated the administration of live probiotic microorganisms, either as single strains or multi-strain formulations, regardless of dose, duration, or route of administration, compared with vehicle or untreated control groups. Studies were required to report at least one of the primary outcomes, including seizure frequency, seizure duration, or markers of neuronal survival or damage. Studies were excluded if they were review articles, meta-analyses, editorials, commentaries, or conference abstracts.

2.3. Study Selection Process

Search results from all databases were imported into EndNote (version 20, Clarivate Analytics) for duplicate removal and study management. Titles and abstracts were independently screened by two reviewers against the eligibility criteria using a standardized screening form. Studies deemed potentially relevant by either reviewer were evaluated in full text. Full-text articles were independently assessed by two reviewers using detailed inclusion and exclusion criteria.

Any disagreements between reviewers were resolved through discussion, and if consensus could not be reached, a third senior reviewer made the final decision. The study selection process was documented using a PRISMA flow diagram, including reasons for exclusion at the full-text stage.

2.4. Data Extraction

Data extraction was performed independently by two reviewers using a standardized, pilot-tested data extraction form developed specifically for this review. Extracted data included: study characteristics (first author name, publication year, study location, study design and experimental duration), animal characteristics (species, sex, age), seizure model (pharmacological agent, electrical stimulation, or other, dose and route of administration for seizure-inducing agents), probiotic intervention (probiotic strain(s) identification and characterization, dose, concentration, and colony-forming units (CFU), route of administration (oral, gavage, drinking water, or other), timing of probiotic administration relative to seizure induction (prophylactic, concurrent, or therapeutic), duration and frequency of probiotic supplementation), and outcome measures (primary outcomes: seizure frequency, seizure duration, secondary outcomes: oxidative stress markers, inflammatory cytokines, behavioral assessments).

2.5. Risk of Bias Assessment

The methodological quality and risk of bias of included studies were independently assessed by two reviewers using the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool, which was designed explicitly for animal intervention studies (Hooijmans et al. 2014). The SYRCLE tool evaluated ten domains. Each domain was rated as "low risk," "high risk," or "unclear risk" based on the information provided in the study reports.

Disagreements between assessors were resolved through discussion, with consultation of a third reviewer if necessary.

2.6. Data Analysis, Heterogeneity, Subgroups, Publication Bias

All quantitative data extracted from eligible preclinical studies were analyzed using meta-analytic techniques. Meta-analyses were conducted using random-effects models (DerSimonian and Laird method), which were selected a priori due to the expected heterogeneity across studies in terms of animal species, probiotic strains, dosing regimens, seizure models, and outcome measurement methods. Results are presented as mean differences (MDs) with corresponding 95% confidence intervals (CIs).

For each outcome (e.g., seizure latency, frequency, severity score, MDA, IL-1β, IL-6, TNF-α, behavioral indices), we calculated pooled effect sizes using mean and standard deviation (SD) or converted standard error of the mean (SEM) to SD using conventional formulas when needed. Where outcomes were reported in different units (e.g., seconds vs. minutes, pg/mg protein vs. pg/mL), we converted them to common units across studies to allow consistent pooling. When outcomes were measured at multiple time points, we extracted data from the last reported day of follow-up to capture the overall effect of the intervention at the end of treatment.

2.7. Software

All statistical analyses and forest plots were performed using R software (version 4.5.1), with the metafor package. Forest plots were generated for each outcome, and only random-effects estimates were displayed (common effect models were excluded).

2.8. Heterogeneity Assessment

Heterogeneity was assessed using the I² statistic and τ² (tau-squared), with I² values of 25%, 50%, and 75% considered low, moderate, and high heterogeneity, respectively. Where high heterogeneity was present (I² > 75%), potential sources of variability were explored qualitatively.

3. Results

3.1. Characteristics and quality assessments of included studies

The first database search produced 226 records. After removing 176 duplicates, 50 records remained for title and abstract screening. Thirteen records were excluded at this stage because they were not related to probiotics and seizure models. Of the remaining 37 full-text articles assessed for eligibility, 13 were excluded due to incomplete data for extraction, lack of probiotic intervention, or use of non-epileptic models. Consequently, 24 preclinical studies were identified as eligible (Fig. 1), and their results are presented in Table 1. All studies were conducted in rodents (rats or mice).

Fig. 1

PRISMA plot for the meta-analysis showing database search details and number of articles included in the study

The included studies were published between 2017 and 2025 and originated from various parts of the world. They also utilized varied models of induced experimental epilepsy, including models induced by pentylenetetrazole (PTZ) (most frequent; more than half of studies) (Bagheri et al. 2019; Tahmasebi et al. 2020; Eor et al. 2021; Eor et al. 2021; Kilinc et al. 2021; Sabouri et al. 2021; Aygun et al. 2023; Ciltas et al. 2023; Thai et al. 2023; Ishii et al. 2024; Shakoor et al. 2024; Zhai et al. 2024; Ali et al. 2025; Matta et al. 2025; Mirzababaei et al. 2025; Tahmasebi et al. 2025), KA (Wang et al. 2022), penicillin (Kızılaslan et al. 2022), and the combination of lithium and pilocarpine (Zubareva et al. 2023), in addition to models of genetic absence seizures (Akkol et al. 2017; Aygun et al. 2022), infantile spasm (Mu et al. 2022; Mu et al. 2022; Mu et al. 2022), and febrile seizure(Nan He 2024).

In all studies, probiotics were administered before and/or after seizure induction. The probiotics tested were diverse, and the most commonly used were Lactobacillus spp. (acidophilus, casei, fermentum, brevis), Bifidobacterium spp. (breve, bifidum, longum), Saccharomyces boulardii, Multi-component formulations (Streptococcus thermophilus HA-110 combined with Lactococcus lactis subsp. lactis HA-136), Synbiotics (combination of probiotic + prebiotic such as inulin (Eor et al. 2021; Wang et al. 2022)). Some studies administered a probiotic and an anticonvulsant drug (e.g., brivaracetam, topiramate, pregabalin). Sample sizes across studies ranged from 24 to 50 animals, with group designs including seizure/epileptic control, probiotic treatment, and some with probiotics alongside antiepileptic drugs (AEDs).

Risk of bias, assessed using the SYRCLE tool, varied across domains. Most studies were judged at low risk for attrition bias (incomplete outcome data), reporting bias (selective outcome reporting), and other sources of bias. However, several domains were frequently rated as “unclear” due to incomplete methodological descriptions, particularly sequence generation, allocation concealment, and blinding of outcome assessors. Performance bias related to random housing was rated low in 15/24 studies and unclear in 9/24, while blinding of caregivers/investigators was low in 8/24 and unclear in 16/24. Random outcome assessment and blinding of outcome assessors were judged to be of low risk in 12/24 studies and unclear in the remaining studies. No domain was consistently rated as high risk of bias, but the large proportion of “unclear” ratings indicates that reporting quality remains a significant limitation of this literature.

3.2. Effects of Probiotic Supplementation on Seizure Activity

The majority of studies have demonstrated the effects of probiotics, especially when combining Lactobacillus and Bifidobacterium strains. These effects have been observed in various models, including PTZ-induced seizures, KA-induced stable epilepsy, penicillin-induced seizures, infantile spasms, and genetic absence epilepsy.

3.2.1. Increased seizure latency

Most studies have reported a significant increase in seizure latency after probiotic administration. These include a delay in the onset of the first myoclonic jerk, an increase in the time to the onset of generalized tonic-clonic seizures, and a slower progression to the fully kindled phase in PTZ models (Tahmasebi et al. 2020; Kilinc et al. 2021; Sabouri et al. 2021zılaslan et al. 2022; Wang et al. 2022; Ciltas et al. 2023; Nan He 2024; Tahmasebi et al. 2025).

3.2.2. Reduced seizure severity and score

Data from several studies demonstrate a significant reduction in seizure severity scores. This reduction includes decreases in the frequency of stage 4–5 seizures, the cumulative seizure score, and the behavioral severity of seizure episodes (Bagheri et al. 2019; Wang et al. 2022; Ciltas et al. 2023; Shakoor et al. 2024).

Some studies have reported that co-treatment with probiotics and AEDs has more favorable effects in reducing seizure severity and score. For example, Thai et al. showed that Lactobacillus johnsonii treatment alone did not significantly mitigate seizure scores. Still, its co-administration with topiramate led to a significant reduction in seizure scores (Thai et al. 2023). Also, in the study by Zhai et al., although probiotics alone reduced seizure severity, this effect was more substantial in the group receiving the combination of probiotics and sodium valproate (Zhai et al. 2024).

3.2.3. Reduced seizure duration and frequency

In the entire dataset, seizure duration was consistently reduced in both acute and chronic models (Wang et al. 2022; Nan He 2024; Zhai et al. 2024; Mirzababaei et al. 2025). Several studies have also reported a reduction in seizure frequency (Akkol et al. 2017; Eor et al. 2021; Sabouri et al. 2021; Aygun et al. 2022zılaslan et al. 2022; Wang et al. 2022; Aygun et al. 2023; Tahmasebi et al. 2025), particularly in STZ, KA, and genetic absence epilepsy models, although the magnitude of the effect in chronic epilepsies has been variable.

In genetic absence epilepsy models, EEG recordings showed that the duration and number of spike-wave discharges (SWDs) were reduced after probiotic treatment (Akkol et al. 2017; Aygun et al. 2022).

Some studies have shown further improvement and reduced seizure frequency with the combination of probiotics and prebiotics. For example, in the study by Eor et al. (2021), the L. fermentum MSK408 strain alone was unable to reduce seizures in the PTZ model. However, the combination of this probiotic with galacto-oligosaccharide (GOS) significantly reduced seizures (Eor et al. 2021).

3.2.4. Synergistic effects in combination with anticonvulsant drugs

Studies using probiotics in combination with anticonvulsant drugs such as diazepam, sodium valproate, topiramate, and brivaracetam (Thai et al. 2023; Shakoor et al. 2024; Zhai et al. 2024) have shown enhanced anticonvulsant effects, including a greater reduction in seizure progression. These results suggest synergistic neuroprotective interactions between probiotics and anticonvulsant drugs.

3.3. Effects of probiotics in modulating neurotransmitters

Following probiotic treatment, increased levels of GABA in brain tissues were reported in some studies (Bagheri et al. 2019; Eor et al. 2021; Ciltas et al. 2023; Matta et al. 2025). This increase in GABA levels, as the most important inhibitory neurotransmitter in the CNS, was concomitant with a decrease in seizure activity. In a study by Thai et al., treatment with the probiotic Lactobacillus johnsonii alone did not affect GABA or glutamate levels in the cerebral cortex. However, following co-treatment with AED (topiramate) and Lactobacillus johnsonii, glutamate was significantly decreased, and the GABA/glutamate ratio was increased, as well (Thai et al. 2023).

An increase in nerve growth factor (NGF) activity, a neuroprotective factor, was also observed, consistent with the anticonvulsant activity of probiotics (Aygun et al. 2022; Aygun et al. 2023). However, probiotics had no significant effect on brain-derived neurotrophic factor (BDNF) levels (Aygun et al. 2022; Aygun et al. 2023) or decreased its activity (Mirzababaei et al. 2025). In addition, an increase in Matrix metalloproteinase (MMP)-9, which is involved in pathological processes including inflammation, was reported following probiotic treatment in the Mirzababaei et al. study (Mirzababaei et al. 2025). These results suggest that probiotics exert protective effects across different seizure models by modulating various neurotransmitter systems and growth factors.

3.4. Effects of probiotic on oxidative stress and antioxidant defense

Almost all studies evaluating markers of oxidative stress found that probiotic administration improved redox imbalance and reduced oxidative stress induced by seizures. Most individual studies reported reductions in oxidative stress markers such as malondialdehyde (MDA) (Bagheri et al. 2019; Mu et al. 2022; Wang et al. 2022; Shakoor et al. 2024; Ali et al. 2025; Mirzababaei et al. 2025), nitric oxide (NO) (Bagheri et al. 2019; Aygun et al. 2022zılaslan et al. 2022; Aygun et al. 2023), total oxidant status (TOS) (Bagheri et al. 2019; Kilinc et al. 2021; Aygun et al. 2023; Ciltas et al. 2023), accompanied by simultaneous increases in antioxidant markers such as superoxide dismutase (SOD) and catalase (CAT) (Shakoor et al. 2024; Ali et al. 2025), reduced glutathione (GSH) (Wang et al. 2022; Mirzababaei et al. 2025), total antioxidant capacity (TAC) (Mirzababaei et al. 2025) or total antioxidant status (TAS) (Ciltas et al. 2023) in brain and liver tissues or plasma. These effects were more evident in multivariate and synbiotic interventions, indicating restoration of redox balance. These results suggest that the antiepileptic and neuroprotective properties of probiotics may be partly explained by their inhibitory effects on neuronal oxidative damage.

3.5. Inflammatory Outcomes

Almost half of the studies reviewed measured pro- and anti-inflammatory cytokines (Eor et al. 2021; Kilinc et al. 2021; Aygun et al. 2022zılaslan et al. 2022; Wang et al. 2022; Aygun et al. 2023; Nan He 2024; Mirzababaei et al. 2025; Tahmasebi et al. 2025), and the extracted data indicated an anti-inflammatory profile for probiotics. There were reduced levels of IL-1β, IL-6, TNF-α, IL-17, and Interferon gamma (IFN-γ) in serum, hippocampus, and cortical extracts treated with both single-strain and multi-strain probiotics. An increased level of IL-10, an anti-inflammatory cytokine, was observed following probiotic treatment in some studies (Mirzababaei et al. 2025; Tahmasebi et al. 2025). These effects were more observable in models with PTZ, KA, and febrile conditions, indicating decreased microglial activation and increased neuronal survival. However, there were also variations across strains and regions, as illustrated by Zubareva et al. (2023), with reduced IL-1β levels in the temporal cortex rather than in the hippocampus (Zubareva et al. 2023). On the other hand, IL-6 levels remained unchanged despite treatment in infantile spasms (Mu et al. 2022). However, the variability in treatment response due to the factors in probiotics, such as dosage and duration, must be considered. Furthermore, combination treatments with multiple strains of Lactobacillus and Bifidobacterium appear to be more effective at reducing inflammation and neuronal injury associated with seizures.

3.6. Behavioral and Cognitive Findings

Findings from behavioral studies indicated that probiotics can mitigate cognitive-emotional changes induced by seizures. Among the primary models used were Morris Water Maze (MWM) and Open Field (OF) tasks. Probiotic (alone or in combination)-treated animals showed shorter escape latencies, and distance moved to reach the hidden platform, and spent more time in the target quadrant (Bagheri et al. 2019; Tahmasebi et al. 2020; Wang et al. 2022; Shakoor et al. 2024; Ali et al. 2025), during MWM tasks. These findings suggest restoration of hippocampal-dependent cognitive function. They also demonstrated higher levels of locomotor activity, more time in the central zone, increased squares crossed, longer grooming duration, and more entries into the center zone during OF tasks (Aygun et al. 2022; Shakoor et al. 2024; Ali et al. 2025; Matta et al. 2025), highlighting reduced anxiety-like behaviors in models of PTZ-induced epilepsy following mixed probiotic treatment.

In addition, some studies showed that probiotic treatment alone or in combination with AED in rats kindled by PTZ increased the percentage of spontaneous alternation in the Y-maze test (Shakoor et al. 2024; Ali et al. 2025; Matta et al. 2025). The novel object recognition test also showed an increase in the discrimination index following probiotic treatment (Shakoor et al. 2024; Ali et al. 2025). Studies examining the Light/Dark test criteria showed increased time spent and the number of entries into the light zone after probiotic treatment, especially when combined with AED (Shakoor et al. 2024; Ali et al. 2025). One study also reported a decrease in immobility time and an increase in swimming time in the forced swimming test following probiotic treatment (Aygun et al. 2022). However, some studies did not replicate the reported effects. In a lithium/pilocarpine model of epilepsy, the administration of B. longum did not significantly affect indices in the OF, elevated plus maze (EPM), social interaction test (SIT), forced swim test (FST), and fear conditioning test (FCT) (Zubareva et al. 2023). Taken together, the cumulative data support the conclusion that supplementation with probiotics, especially multi-strain Lactobacillus-Bifidobacterium, can have a positive effect on behavioral parameters in experimental models of epilepsy. These effects may be due to the combined actions of inhibiting neuroinflammation, restoring redox balance, and regulating synaptic plasticity, which, together, contribute to the fundamental role of the gut-brain axis in both the pathogenesis of epilepsy and cognitive deterioration induced by epileptic seizures.

4. Findings of the Meta-Analysis

4.1. Effects of Probiotics on Seizure-Related Outcomes

Six studies assessed seizure severity scores. The pooled analysis using a random-effects model demonstrated a statistically significant reduction in severity scores in the probiotic group (MD = − 1.08; 95% CI: −1.39 to − 0.76; p < 0.001), with low heterogeneity (I² = 27.3%, τ² = 0.04), suggesting consistent findings across studies (Fig. 2a). Five studies measured seizure duration. The meta-analysis showed a significant decrease in seizure duration in the probiotic-treated groups (MD = − 23.19; 95% CI: −35.56 to − 10.82; p < 0.001). However, heterogeneity was extremely high (I² = 96.5%, τ² = 161.42), suggesting variability in methodology across the included studies (Fig. 2b). Twelve studies evaluated seizure latency after probiotic supplementation in animal models. The random-effects model revealed a significant increase in latency in the probiotic groups compared to controls (MD = 22.09; 95% CI: 10.52 to 33.67; p < 0.001), suggesting a delayed onset of seizures. However, heterogeneity was substantial (I² = 94.2%, τ² = 237.67) (Fig. 2c). Five studies reported seizure frequency data. The meta-analysis indicated a non-significant reduction in seizure frequency (MD = − 4.53; 95% CI: −10.16 to 1.10; p > 0.05), although a strong downward trend was observed. Moderate heterogeneity was present (I² = 57.7%, τ² = 16.62) (Fig. 2d). Taken together, these results indicate that probiotics are most consistently effective in prolonging seizure latency and reducing severity and duration, but effects on seizure frequency remain unclear. The low heterogeneity in severity score outcomes strengthens confidence in this finding, while the high variability in latency and duration results warrants further subgroup exploration.

Fig. 2

Forest Plot of Pooled Effects of Probiotic Supplementation on Seizure-Related Outcomes in Preclinical Models: A Random-Effects Meta-Analysis

4.2. Effects of Probiotics on Inflammatory Markers and Oxidative Stress

Five studies assessed IL-1β levels in pg/mg protein or pg/mL. The meta-analysis using a random-effects model revealed a significant reduction in IL-1β levels following probiotic supplementation compared to control groups (MD = − 45.21; 95% CI: −86.19 to − 4.22; p < 0.05). Substantial heterogeneity was observed among studies (I² = 89.1%, τ² = 1431.91) (Fig. 3a). Meta-analysis of TNF-α levels from five studies indicated a significant reduction in the probiotic group (MD = − 17.93; 95% CI: −33.46 to − 2.41; p < 0.05). The model demonstrated extremely high heterogeneity (I² = 99.7%, τ² = 286.19), suggesting variability in the magnitude of the effect across different models or strains (Fig. 3b). Eight studies evaluated IL-6 levels. The meta-analysis revealed a significant decrease in IL-6 following probiotic intervention (MD = − 0.69; 95% CI: −1.21 to − 0.17; p < 0.05). However, the between-study heterogeneity remained considerable (I² = 97.3%, τ² = 0.3848), necessitating cautious interpretation (Fig. 3c). A total of 8 studies reported malondialdehyde (MDA) concentrations as a marker of oxidative stress. The pooled effect size from the random-effects model showed a non-significant trend toward reduction in MDA (MD = − 6.47; 95% CI: −13.65 to 0.72; p = 0.076), with high heterogeneity across studies (I² = 98.9%, τ² = 40.19) (Fig. 3d). Collectively, these findings suggest that probiotic supplementation attenuates markers of oxidative stress and inflammation in experimental models of seizure, most consistently for IL-1β, IL-6, and TNF-α. High heterogeneity across studies may be attributed to differences in probiotic strains, dosages, seizure models, and outcome assessment techniques.

Fig. 3

Forest Plot of Pooled Effects of Probiotic Supplementation on inflammatory and oxidative Outcomes in Preclinical Models: A Random-Effects Meta-Analysis

4.3. Effects of Probiotics on Behavioral Outcomes

Five studies evaluated escape latency in the Morris Water Maze (MWM), a spatial learning and memory task. The random-effects model demonstrated a statistically significant reduction in escape latency in the probiotic group (MD = − 10.29; 95% CI: −20.45 to − 0.14; p < 0.05), indicating improved spatial learning performance. However, heterogeneity was substantial (I² = 95.9%, τ² = 118.19) (Fig. 4a). The same five studies also reported time spent in the target quadrant. The random-effects meta-analysis showed no significant difference between groups (MD = 6.68; 95% CI: −9.72 to 23.08; p = 0.37). The heterogeneity was extremely high (I² = 98.5%, τ² = 277.08), indicating substantial variability across studies in the measurement of retention memory (Fig. 4b).

Fig. 4

Forest Plot of Pooled Effects of Probiotic Supplementation on Behavioral Outcomes in Preclinical Models: A Random-Effects Meta-Analysis

Six studies assessed center entries in the open field test as a proxy for anxiety-like behavior. The random-effects model showed a significant increase in center entries in the probiotic group (MD = 17.28; 95% CI: 10.77 to 23.79; p < 0.001), reflecting a reduction in anxiety-like behavior. Heterogeneity was low (I² = 24.6%, τ² = 16.13), suggesting relatively stable effects across studies (Fig. 4c). Four studies examined locomotor activity via total distance traveled. The pooled effect size was not statistically significant (MD = 0.78; 95% CI: −0.48 to 2.04; p = 0.2). Heterogeneity was negligible (I² = 0%, τ² = 0), indicating consistent results across studies (Fig. 4d). These findings support the potential anxiolytic and cognitive-enhancing effects of probiotics in seizure models, particularly as seen in MWM escape and OFT center entries. The non-significant findings in MWM target time and OFT distance suggest that these effects are more specific to emotional and memory-related domains, rather than general locomotion.

5. Discussion

The findings of this systematic review and meta-analysis suggest that probiotics exert their protective and anticonvulsant effects through multiple pathways that are directly involved in the pathophysiology of seizures. Probiotics significantly increased seizure latency and reduced seizure severity and duration, with a non-significant but directionally favorable trend toward reduced seizure frequency. These effects were observed across different seizure models and indicated increased seizure threshold and decreased neuronal network excitability. This pattern was consistent across most models used, suggesting that the effects of probiotics were model-independent and largely consistent across models.

Probiotics and prebiotics can act through several pathways, including strengthening the intestinal barrier, reducing pathogen and lipopolysaccharide (LPS) penetration into the bloodstream, reducing systemic inflammation, producing beneficial metabolites, such as SCFAs, and regulating immunity (Ansari et al. 2023). In recent years, the role of the gut-brain axis in neurological diseases, including epilepsy, has attracted widespread attention. Human studies have shown that patients with epilepsy, especially those with drug-resistant epilepsy, have significant disturbances in the diversity and composition of the gut microbiome. This pattern of dysbiosis is often associated with higher levels of systemic inflammation and increased neuronal excitability (Chatzikonstantinou et al. 2021; Mousavi et al. 2025). The results of the present study are consistent with these reports and suggest that restoring microbiome balance through probiotics can have significant protective effects on neural networks.

Another pathway related to the factors evaluated is the regulation of neurotransmitters, which was investigated in the present studies through the GABA/glutamate ratio. A disturbance in the ratio of excitatory to inhibitory neurotransmitters is one of the most critical factors in the pathogenesis of seizures. An increase in the GABA-to-glutamate ratio is associated with reduced neuronal network excitability and increased seizure threshold (Perucca et al. 2023). Species such as Lactobacillus brevis, L. helveticus, and L. rhamnosus can activate glutamate decarboxylase, increase GABA production, or alter the expression of GABA-A receptors (Bravo et al. 2011; Hasegawa et al. 2020; Icer et al. 2024). This effect is significant in PTZ models that induce seizures by blocking GABA-A receptors. Preclinical studies have shown that probiotics increase seizure latency and reduce seizure severity by increasing GABA and decreasing glutamate. In addition, SCFAs also reduce excitotoxicity by modulating vagal neurons and regulating hippocampal network activity (Ciltas et al. 2023; Matta et al. 2025). SCFAs, especially butyrate and propionate, reduce the expression of inflammatory genes and activate protective pathways by inhibiting histone deacetylases (HDACs), causing epigenetic changes and activating GPR41/43 receptors. These metabolites also strengthen the intestinal and blood-brain barriers by increasing tight junction proteins such as claudin-5 and occludin, which prevent the entry of inflammatory cytokines and metabolites, including LPS, into the brain. Reduced SCFA production due to microbial dysbiosis can weaken the intestinal barrier, lead to the leakage of LPS and other inflammatory products into the blood and brain, and predispose to neuroinflammation, increasing neuronal sensitivity and seizure susceptibility. Restoring SCFA production by probiotics has significant anti-inflammatory and neuroprotective effects (Zhang et al. 2022; Shokr et al. 2025).

Increased proinflammatory cytokines in various models of epilepsy have been shown to lower the seizure threshold, increase blood–brain barrier permeability, activate microglia, and increase neuronal excitability (Viviani et al. 2003; Roseti et al. 2015; Chen et al. 2021; Geng et al. 2025). In such situations, reducing these cytokines could directly reduce the neural network's sensitivity. Studies in this review showed that probiotics significantly reduced IL-1β, TNF-α, and IL-6 in the hippocampus and cerebral cortex and increased IL-10, an effect likely mediated by inhibition of the TLR4/NF-κB pathway and reduced microglial activation (Wang et al. 2022). These effects are also consistent with recent evidence that probiotics and prebiotics inhibit the TLR4, NF-κB, and NLRP3 inflammasome pathways and reduce neuroinflammation by increasing SCFA production. They reduce TNF-α and IL-1β secretion in microglia, reduce ROS, NO, and iNOS production, and restore mitochondrial metabolism, thereby maintaining microglial protection (Wang et al. 2022; Caetano-Silva et al. 2023; Kundu et al. 2023; Yang et al. 2024; Cao et al. 2025). It has also been reported that the use of probiotics as an adjunctive therapy in drug-resistant epilepsy can reduce seizure frequency and improve patients' quality of life by modulating inflammatory pathways and restoring microbiome balance (El-Sharkawy et al. 2024). Therefore, as discussed in this review, reducing neuroinflammation is a key mechanism underlying the anticonvulsant effects of probiotics.

Increased MDA as an indicator of lipid peroxidation and decreased SOD, GSH, and CAT have been repeatedly reported in many epilepsy models, suggesting a role for oxidative stress in facilitating the onset and persistence of seizures (Shin et al. 2011; Łukawski and Czuczwar 2023; Yilgor and Demir 2024). In the studies reviewed, probiotics generally improved redox balance, with most individual experiments reporting reduced MDA and increased antioxidants such as SOD, TAC, and GSH. In our meta-analysis, the pooled effect on MDA showed a non-significant trend toward reduction, but the direction of effect across studies was consistent with an antioxidant profile. This effect is likely mediated by anti-inflammatory and antioxidant microbial metabolites, such as SCFAs (such as butyrate), which stimulate the production of antioxidant enzymes through activation of the Nrf2/ARE pathway (Yang et al. 2024). Restoring oxidative balance reduces neuronal damage, preserves neuronal membrane function, and ultimately increases seizure threshold.

On the other hand, the behavioral and cognitive effects reported in existing studies-including improved performance in MWM and OFT- can be explained by mechanisms that reduce inflammation, improve redox balance, and regulate neurotransmitter function. Neuroinflammation and oxidative stress are two critical factors in memory impairment and post-seizure anxiety (Pearson et al. 2015; Elhady et al. 2024; Khan et al. 2024). Moreover, reducing these factors can improve hippocampal function and network connectivity. The improvement in behavioral and cognitive function following probiotic treatment is mediated through mechanisms including increased BDNF levels, increased hippocampal neurogenesis, and regulation of synaptic activity in the CA1 and DG pathways (Arulsamy et al. 2020; Aygun et al. 2023).

Despite these synergistic mechanisms, heterogeneity across several outcomes (e.g., seizure latency, duration, IL-6, TNF-α, MDA, and some behavioral measures) was substantial to extreme (I² often > 75%), likely reflecting differences in probiotic strains, doses, timing, seizure models, animal characteristics, and outcome assays. We had planned subgroup and meta-regression analyses, as well as formal tests of publication bias. Still, each meta-analyzed outcome included fewer than 10 studies, limiting the feasibility and reliability of these approaches. This underscores the need for future preclinical studies with standardized protocols, transparent reporting, and sufficient sample sizes to enable robust stratified and bias assessments.

6. Conclusion and future research directions

The findings of this study suggest that probiotics exert anticonvulsant and neuroprotective effects in preclinical epilepsy models, mainly by modulating the gut–brain axis, reducing neuroinflammation, improving neurotransmitter balance, and influencing oxidative stress and neuronal metabolism. These effects were most consistent for seizure latency, severity, and inflammatory markers (IL-1β, IL-6, TNF-α), whereas evidence for seizure frequency and MDA was less conclusive.

Given the marked heterogeneity and the exclusive reliance on rodent data, these results should be viewed as hypothesis-generating rather than directly applicable to clinical practice. Even so, they support further evaluation of specific probiotic formulations as adjunctive candidates in epilepsy, ideally through rigorously designed preclinical studies with standardized strains and dosing, followed by well-powered clinical trials incorporating cognitive, electrophysiological, microbiome, and metabolomic outcomes.

Declarations

Competing Interests:

None.

Funding:

None.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT (GPT-3.5), an AI language model developed by OpenAI, accessed via the free web version in order to improve the readability and language of the manuscript.

After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Data availability statements:

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary file. The data are available from the corresponding author upon reasonable request.

Author's Contributions

L.S. designed, conceived, and planned the study, analyzed data, calculated statistics, interpreted the data, and drafted and revised the manuscript for intellectual content. E.H. designed, acquisition of data, interpreted the data, and drafted and revised the manuscript for intellectual content. R.H. acquisition of data, interpreted the data, and revised the manuscript for intellectual content. S.H. acquisition of data and revised the manuscript for intellectual content.

Author, year	Gender/species/strain/weight or age	Animals per group (SE/treat)	SE model	Type of probiotic	Protocol of treatment	Outcomes
Akkol et al. 2017	Male/ rat/ WAG / 5–12 month	5/4	Genetic Absence Epilepsy	Multi strain	1 month	Cumulative Duration spike-and-wave discharges
Ali et al. 2025	Male/mice/BALB/ 12–27 g	8/6	PTZ	Multi strain	21 days before SE	OFT, MWM, Light and dark box test, Y-maze, SST/ MDA, catalase, SOD
Aygun et al. 2022	Male/ rat/ Wistar/ adult	7/7	PTZ	Multi strain	6 weeks before SE	ECoG/ TNF-α, Il-6, TOS, NO, BDNF, NGF, Sox2
Aygun et al. 2022	Male/ rat/WAG/6–7 months	7/7	Genetic Absence Epilepsy	Multi strain	30–46 days	ECoG/ OFT, FWT/BDNF, NGF, Sox2, TNF-α, Il-6, NO
Bagheri et al. 2019	Male/ rat/ Wistar/ old	8/8	PTZ	Multi strain	21 days before SE model	MWM/MDA, NO, TOS, GABA
Ciltas et al. 203	Male/ rat/ Wistar/ adults/ 230-260g	6/6	PTZ	Multi strain	21 days before SE model	Seizure severity score/GABA, Glu, TAS, TOS
Eor et al.2021	Male/ mice/ 3-week-old	5/5	PTZ	Multi strain	once a day for 8 weeks before SE model	Seizure frequency, latency/ GABA, Glu
Eor et al. 2021	Male/ mice/3-week-old	5/5	PTZ	One- strain	once daily for 4 weeks before SE model	Seizure severity score, latency, seizure frequency/ TNF-α, GABA
Author, year	Gender/species/strain/weight or age	Animals per group (SE/treat)	SE model	Type of probiotic	Protocol of treatment	Outcomes
Nan He et al, 2024	Male/ mice/SPF/ 22–24 g	6/6	Febrile seizure	One-strain	7 days before SE	Il-β, il-6, TNF-α, iNOS, GFAP
Ishii et al, 2024	Male/ mice/ C57BL6/7–8 weeks	6–9/6–9	PTZ	One- strain	15 days with PTZ injection	ILK, neuropsin, MMP-9, BDNF, P-Akt
Kilinc et al.2021	Male/ rat/ Wistar/ 4 weeks/ 50–70 g	12/12	PTZ	Multi- strain	60 days before SE model	Latency, duration, severity score/ Il-β, Il-6, Il-17, IFN, TOS, TAS
Kızılaslan et al. 2022	Male/ rat/ Wistar	7/7	Penicillin-induced focal seizure	Multi-strain	30 days before induced	Seizure frequency, latency, amplitude/ Il-6, TNF-α, NO
Matta et al. 2025	Male/ rat/ Wistar/ 6 weeks/150–200 g	6/6	PTZ	One- strain with 3 different doses	3 weeks before induced	Seizure severity score/ TNF-α, Il-6, IFN-γ, CRP, GABA/ OFT, Y-maze
Mirzababaei et al. 2025	Male/ rat/ Wistar/ 200–240 g	9/9	PTZ	One-strain	57 days before induced	Latency, duration/ Il-1β, Il-6, Il-10, BDNF, MMP-9, MDA, GSH, TAC
Mu et al. 2022	Both/ rat/ Wistar/ post neonatal	7/7	Infantile Spasms	Multi-strain	1 day pretreatment and 4 days posttreatment	MDA, IL-18, IL-1β, IL-6
Mu et al. 2022	Rat / Wistar/ neonatal	6/6	Infantile spasms	Multi-strain	5 days after induction	Mitochondrial respiration assays, NAAG
Sabouri et al. 2021	Male/ mice/ NMRI/ 25–30 g	6/6	PTZ	Multi-strain	14 days before induction	Duration, latency,

Author, year	Gender/species/strain/weight or age	Animals per group (SE/treat)	SE model	Type of probiotic	Protocol of treatment	Outcomes
Shakoor et al. 2024	Male/mice/ BALB/ 18–25 g	10/10	PTZ	Multi-strain	21 days before induction	Seizure severity score, / MDA, SOD, catalase/ OFT, Y-maze, NOR MWM, Light/Dark test
Tahmasebi et al. 2020	Male/ rat/ Wistar/ 120–150 g	8/8	PTZ	Multi-strain	10 weeks: (6 weeks before starting PTZ kindling, and 4 weeks afterwards	Seizure severity score, latency/ MWM,
Tahmasebi et al. 2025	Male/ rat/ Wistar/ 180–220 g	10/10	PTZ	Multi-strain	8 weeks: 4 weeks before seizure induction and 4 weeks afterwards	Latency, seizure severity score, frequency/ IL-1β, IL-6, IFN-G, IL-10
Thai et al. 2023	Male/ mice/ C57BL/6J/ 9 weeks old	6–8/6–8	PTZ	One-strain	7 days before induction then continued during kindling in treatment groups	Mortality rate/ GABA/Glu ratio
Wang et al. 2022	Male/rat/ Wistar/ 280-300g	NA	Kainic acid	Multi- strain	From day of SE induction for the duration of the experiment (until day 28)	Seizure score, frequency of seizure, duration, /MDA, OHdG, GSH, IL-1β, IL-6, TNF-α, IFN-γ/MWM
Zhai et al. 2019	Male/ mice/ Kunming/18–22 g	10/10	PTZ	Multi-strain	30 days before to PTZ	Duration. Latency, seizure severity score
Zubareva et al. 2023	Male/rat/Wistar/ 7-week-old	16–20/16–20	Pilocarpine	One-strain	30 days after to pilocarpine	Mortality rate/ IL-1β, IL1rn/Pparg gene/OFT, EPM, FST, FCT

Graphical Abstract

The potential effects of probiotics in seizure models

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