Effects of Polydatin on Pentylenetetrazol-Induced Seizures in Zebrafish Larvae

Abstract

Background/Objectives: Epilepsy is a common neurological condition characterized by the occurrence of a seizure. It affects around 50 million individuals worldwide, and despite the large quantity of anti-seizure medications available, 30% of epileptic patients still suffer from seizures. Therefore, it is necessary to find new therapeutic options. Interestingly, polydatin has shown promising effects on epilepsy treatment due to its antioxidant and anti-inflammatory properties. Thus, this study aimed to evaluate the effects of polydatin (200, 300, and 400 µM) on a pentylenetetrazol (PTZ)-induced seizure model in wild-type zebrafish (Danio rerio) larvae. Methods: Seizure-like behavior, cell death, reactive species (RS) production, and lipid peroxidation were analyzed. Results: Pre-treatment with polydatin at 200 and 300 µM did not have a significant impact on seizure occurrence and the behavior of animals exposed to PTZ. Diazepam decreased seizure occurrence and increased the latency to achieve each seizure stage. Exposure to PTZ increased the swimming activity, and this effect was suppressed by diazepam but not by polydatin. PTZ exposure increased the RS production, which was significantly attenuated by polydatin at 400 µM and DMSO. Cell death and lipid peroxidation were not changed when compared to the experimental groups. Conclusions: Only the experimental positive control (diazepam) showed anti-seizure effects. Therefore, we failed to observe any anti-seizure effects of polydatin using a zebrafish experimental model. However, we cannot rule out its effects in other experimental models and different treatment protocols.

1. Introduction

Epilepsy is a chronic neurological condition that affects approximately 50 million people worldwide, representing a significant challenge for healthcare systems [1]. It is characterized by recurrent seizures, which are transient events marked by abnormal neuronal activity in the brain. Seizures are accompanied by repetitive behavioral changes that reflect the underlying neural mechanisms of the disease [1,2,3].

The pharmacological treatment of epilepsy is symptomatic and aims to reduce the frequency and severity of seizures through the modulation of voltage-gated ion (sodium, calcium, and potassium) channels, enhancement of γ-aminobutyric acid (GABA)-mediated inhibition, and inhibition of the synaptic excitation mediated by ionotropic glutamate receptors. Unfortunately, despite the availability of around 40 therapeutic options, about 30% of individuals with epilepsy are unable to achieve satisfactory seizure control. Furthermore, approximately 80% of individuals experience adverse effects associated with these drugs. Therefore, patients with epilepsy have their quality of life impaired [4].

Interestingly, advances in pharmaceutical research have highlighted the potential of natural products in the discovery and development of pharmaceutical inputs. Many drugs are derived from substances extracted from medicinal plants, demonstrating their importance in the development of innovative therapies [5]. Among natural products, plant-derived polyphenols have been studied for their antioxidant, anti-inflammatory, and neuroprotective properties, being explored as therapeutic alternatives for neurological diseases such as epilepsy [6].

Resveratrol, a widely studied plant-derived polyphenol, is known for its multiple therapeutic properties, including antioxidant, anti-inflammatory, and neuroprotective actions, which are attributed to its ability to neutralize free radicals and modulate inflammatory and apoptotic pathways [7,8,9,10]. Studies have demonstrated the beneficial effects of resveratrol in the management of convulsive-type seizures and neuroprotection. Resveratrol was able to prolong the latency period of seizures and reduce their severity in adult zebrafish exposed to N-methyl-D-aspartic acid [11]. Micronized resveratrol shows promising effects in a seizure model in zebrafish and signalizes an important advance in epilepsy treatment [12,13]. Furthermore, it demonstrated anticonvulsant activity without causing renal or hepatic damage in a model of lithium/pilocarpine-induced status epilepticus in rats [14].

Despite its promising therapeutic properties, the clinical application of resveratrol is limited by its low bioavailability and rapid metabolism [15]. In this context, polydatin (Figure 1), a resveratrol glycoside, emerges as a promising alternative. Presenting a glycosylated structure, polydatin offers greater stability and better bioavailability [16]. Studies indicate that polydatin also possesses significant neuroprotective, anti-inflammatory, and antioxidant properties, with the potential to cross the blood–brain barrier and protect against oxidative stress and inflammation [17]. Polydatin has shown potential in preventive and/or therapeutic actions for a variety of neurological conditions. Its benefits are attributed to its ability to mitigate oxidative stress through various mechanisms, including the modulation of enzymatic antioxidants (superoxide dismutase, catalase, and glutathione peroxidase) and non-enzymatic antioxidants (reduced glutathione). It also modulates key antioxidant pathways, including the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and sirtuin 1 [18]. Despite the promising characteristics of polydatin, its effects on epilepsy and seizures have not been sufficiently explored.

In this context, given the complexity of epilepsy and the limitations of current therapies, as well as the pharmacological potential of polyphenols, the present study aimed to investigate the effects of polydatin in a model of seizure-like symptoms induced by pentylenetetrazol (PTZ) in zebrafish larvae. To this end, the effects of polydatin on behavioral parameters, cell death, and oxidative stress were evaluated.

2. Materials and Methods

2.1. Chemicals

Pentylenetetrazol (CAS number: 54-95-5), polydatin (CAS number: 65914-17-2), acridine orange (CAS number: 65-61-2), 2′,7′-dichlorofluorescein diacetate (CAS number: 2044-85-1), MS-222 (CAS number: 886-86-2), and methylcellulose (CAS number: 9004-67-5) were obtained from Sigma-Aldrich^® (St. Louis, MO, USA). The stock solution of polydatin was prepared in dimethyl sulfoxide (CAS number: 67-68-5) and stored at −20 °C. Diazepam (CAS number: 439-14-5) was used as a positive control. The other reagents used in the experiments were of analytical grade, including thiobarbituric acid (CAS number: 504-17-6), butylated hydroxytoluene (CAS number: 128-37-0), acetic acid (CAS number: 64-19-7), sodium dodecyl sulfate (CAS number: 151-21-3), 1,1,3,3-tetramethoxypropane (CAS number: 102-52-3), butanol (CAS number: 71-36-3), ethanol (CAS number: 64-17-5), CuSO₄·5H₂O (CAS number: 7758-99-8), Na₂CO₃ (CAS number: 497-19-8), NaOH (CAS number: 1310-73-2), and buffer (100 mM Tris-HCl, 2 mM EDTA, 5 mM MgCl₂·6H₂O). E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄; pH 7.2) was used to dissolve the solutions.

2.2. Animals

The progenitors of the zebrafish larvae (Danio rerio) wild-type were obtained from Delphis Aquários e Pet Shop (Porto Alegre, RS, Brazil) and maintained in an aquarium system that uses non-chlorinated water, with recirculation and automated control of physicochemical parameters (Altamar Sistemas Aquáticos, Jacareí, SP, Brazil). The parameters included a temperature of 28 ± 1 °C, conductivity of 500 ± 50 μS/cm, pH between 7.2 ± 0.5, and a light/dark cycle (12/12 h). The animals were housed in 10 L aquariums with a density of 2 fish per liter. Feeding was provided three times a day, consisting of commercial flake fish food (Supervit^®,Porto Alegre, RS, Brazil) and Artemia salina (BioArtemia, Grossos, RN, Brazil) [19,20,21].

Zebrafish larvae were obtained through breeding adult zebrafish in a ratio of 2 males to 1 female. At the end of the day, screen traps were placed at the bottom of the aquarium to prevent predation of the eggs and were removed after the start of the light cycle the following morning. The fertilized eggs were collected and transferred to Petri dishes, with a ratio of one animal/mL of E3 medium for a total of 25 embryos per dish. The eggs were then kept in an incubator at a constant temperature of 28 °C with a light/dark cycle (12/12 h). The viability of the animals was monitored daily, and the E3 medium was replaced until the start of the experiment.

The handling and care of the animals were carried out in accordance with the Brazilian Guidelines for the Practice of Animal Care and Use for Scientific and Educational Purposes [22]. All the experiments were approved by the Animal Ethics Committee (CEUA) of the Federal University of Rio Grande (FURG) under the number 23116.007121/2023-85.

2.3. Experimental Design

Healthy animals without malformations were included in the study, and the sample size was determined based on the existing literature, considering the behavioral outcomes. Zebrafish larvae at 6 days post-fertilization (dpf) were aleatorily selected and individually placed in a 96-well plate, where larval treatments were performed by immersion. To minimize potential disturbances, the animals were placed in 96-well plates following an alternating pattern, with occupied and empty wells. This arrangement ensured that each animal was positioned in alternating wells, providing the necessary spacing to reduce unwanted interactions. Polydatin was applied to the animals 24 h prior to PTZ exposure at three concentrations: 200, 300, and 400 µM, according to the study by Schimith et al. (2024) [23]. The vehicle-treated group received 0.61% DMSO, which corresponds to the concentration found in the highest polydatin concentration. The other concentrations of polydatin were prepared using lower concentrations of DMSO. Therefore, to minimize the number of animals used, only the highest concentration was selected as the control. Diazepam (DZP, 75 µM) was dissolved in the E3 medium and used as a positive control, administered 2 h prior to PTZ exposure [13]. The control group received only the E3 medium.

At 7 dpf, the treatments were renewed and were followed by exposure to PTZ at a concentration of 10 mM. After exposure, behavioral analysis was performed over a 60 min period, evaluating the occurrence and latency to reach stage I, stage II, and stage III of the seizure [24]. Subsequently, analyses were conducted to assess cell death, reactive species production, and lipid peroxidation using samples collected after the 60 min PTZ exposure [25,26,27], as shown in Figure 2.

2.4. Analyses of Seizure-like Behavior

After the pre-treatments, 35 zebrafish larvae per experimental group (n = 7 animals per group, 5 independent replicates) were placed for acclimatization in the dark for 20 min in a system for acquiring images and activity and locomotion data patterns (DanioVision, Noldus^®, Wageningen, The Netherlands). In the diazepam-treated group, 30 animals were used (n = 6 animals, 5 independent replicates). Subsequently, the animals were exposed to 10 mM PTZ, and their behavior was recorded using the DanioVision observation camera (Basler acA 1300-60, Ahrensburg, Germany) for 60 min. Locomotor activities were assessed in the 96-well plates, and the parameters of total distance, average velocity, and mobility were recorded and analyzed using the Ethovision XT software version 11.5 (Noldus^®, Wageningen, The Netherlands) [28].

2.5. Seizure Occurrence and Latency Scoring

The occurrence and latency of the first seizure were recorded for stages 1, 2, and 3 over a maximum period of 10 min. The quantification of seizures was analyzed in a blinded manner by 2 experimenters. Stage 1 was characterized by a marked increase in swimming activity, indicating hyperactivity. In Stage 2, the fish exhibited rapid circular swimming, resembling a whirlpool. Finally, Stage 3 consisted of brief clonus-like seizures accompanied by loss of posture, evidenced by lateral body tilting and immobility lasting at least 3 s [24].

2.6. Cell Death Analysis

Cell death analysis was performed using the fluorescent dye acridine orange (AO). For the analysis, 18 larvae per experimental group (n = 6 animals per group, 3 independent replicates) were incubated in the dark in an AO solution at a concentration of 5 µg/L at 28 °C for 30 min. The larvae were then washed with the E3 medium and anesthetized with tricaine (168 mg/mL). Subsequently, the larvae were individually placed on histological slides containing 3% methylcellulose for immobilization and image capture. Pyknotic points were observed using an inverted fluorescence microscope (Olympus IX81, Olympus, New York, NY, USA). The analysis was conducted with emission/excitation wavelengths of 475/530 nm, and the exposure time for image capture was 77 ms. Data were acquired using the CellSens Dimension software version 1.6 (Tokyo, Japan) and subsequently analyzed in ImageJ version 1.54g (Bethesda, MD, USA) [25]. The animals were then euthanized by immersion in liquid nitrogen.

2.7. Measurement of Reactive Species (RS) Production

The measurement of RS production was performed using the fluorescent dye 2′,7′-dichlorofluorescindiacetate (DCFH-DA). For the measurement, 18 larvae per experimental group (n = 6 animals per group, 3 independent replicates) were incubated in the dark in a DCFH-DA solution (4.85 µg/mL) at 28 °C for 30 min. After incubation, the larvae were washed with the E3 medium and anesthetized with tricaine (168 mg/mL). Subsequently, the larvae were placed individually on histological slides containing 3% methylcellulose for immobilization and image capture. Fluorescence intensity was visualized using a fluorescence microscope (Olympus BX51, Tokyo, Japan). Observations were made with emission/excitation wavelengths of 460/520 nm, and the exposure time for image capture was 72 ms. Data were acquired using the CellSens Entry software version 1.6 (Tokyo, Japan) and subsequently analyzed in ImageJ version 1.54g (Bethesda, USA) [26]. The animals were then euthanized by immersion in liquid nitrogen.

2.8. Lipoperoxidation

Lipoperoxidation was assessed using the thiobarbituric acid reactive substances (TBARS) method by fluorimetry, as described by Oakes and Kraak (2003) [27]. The animals were then euthanized by immersion in liquid nitrogen, and four pools were prepared for each experimental group, each containing 21 larvae. In brief, the larvae were homogenized in 100 mM Tris-HCl, 2 mM EDTA, and 5 mM MgCl₂·6H₂O, the pH adjusted to 7.75, and then centrifuged at 10,000,000× g for 20 min at 4 °C. The supernatant was separated and incubated with 1.407 mM butylated hydroxytoluene (BHT), 20% acetic acid, 0.8% thiobarbituric acid, water, and 8.1% sodium dodecyl sulfate (SDS) and incubated at 95 °C for 30 min. After cooling, 100 µL of water and 500 µL of n-butanol were added, and the samples were centrifuged at 10,000,000× g for 10 min at 4 °C. The organic phase was measured at an emission/excitation wavelength of 520/580 nm using a microplate reader (Victor 2 PerkinElmer, Waltham, MA, USA). The concentration of malondialdehyde (MDA) was quantified based on a standard curve of 1,1,3,3-tetraethoxypropane (TMP). Total protein was quantified using the method of Lowry et al. (1951) [29]. The results were expressed as nmol of MDA per mg of protein.

2.9. Statistical Analysis

To ensure data consistency, outlier identification was performed using the ROUT test (Q = 1%). Data distribution was assessed using the Shapiro–Wilk normality test. For data that followed a normal distribution, a one-way ANOVA was applied, followed by Tukey’s post-hoc test for multiple comparisons, with results expressed as mean ± standard error of the mean (SEM). For non-parametric data, the Kruskal–Wallis test was used, followed by Dunn’s test, and the results were presented as median ± interquartile range (IQR). Differences between groups were considered statistically significant for p-values < 0.05.

3. Results

3.1. Effects of Polydatin on Seizure-like Occurrence and Latency

The results of the occurrence and latency of seizures in stages I, II, and III are shown in Figure 3 and Figure 4. Considering the latency for stages I, II, and III, no differences were observed between the groups pre-treated with polydatin at concentrations of 200, 300, and 400 µM and the group receiving DMSO + PTZ. The larvae exposed to PTZ showed a higher incidence of reaching the tonic–clonic seizure stage (III) compared to the control group. The control and diazepam groups increased the latency to reach all three stages (p < 0.0001) compared to the PTZ group.

3.2. Effects of Polydatin on Seizure-like Behavior

The data from the present study demonstrated that PTZ increased the total distance, average speed, and mobility time of the animals compared to the control group, confirming its ability to induce convulsive behavior (p < 0.05). Pre-treatment with polydatin at concentrations of 200, 300, and 400 µM showed no differences when compared to animals that received DMSO plus PTZ. The positive control, diazepam, reduced the behavioral parameters of the larvae (p < 0.05) compared to the PTZ group.

To complement the locomotor analysis, a tracking graph and heatmap were employed for each group, providing a more dynamic visualization of the experimental data. The tracking allows for a detailed monitoring of the sample behavior over time, enabling the recognition of the displacement path of the groups. On the other hand, the heatmap provides a graphic that highlights the areas where individuals spend more time, as shown in Figure 5. In this representative figure, it can be observed that the animals exposed to PTZ (B) had greater locomotor activity compared to the control group (A), as evidenced by the reduction in warm color areas. The diazepam group (E) was effective as a positive control for the experimental model.

3.3. Effects of Polydatin on Cell Death

Figure 6 shows the data from the acridine orange (AO) staining. It was observed that exposure to PTZ led to an increase in the number of pyknotic nuclei compared to the control group (p < 0.05). In contrast, pre-treatment with polydatin at concentrations of 200, 300, and 400 µM did not result in significant changes. The positive control, diazepam, reduced the number of pyknotic nuclei compared to the PTZ-exposed group, indicating a protective effect against cell death (p < 0.05).

3.4. Effect of Polydatin on the Production of RS

The data obtained from the DCFH-DA assay showed that exposure to PTZ resulted in an increase in RS production compared to the control group (p < 0.05). Pre-treatment with polydatin at a concentration of 400 µM and the DMSO + PTZ showed no differences between them. Polydatin concentrations of 200 and 300 µM exhibited an increase in RS production compared to the DMSO + PTZ (p < 0.05), as illustrated in Figure 7.

3.5. Effects of Polydatin on Lipid Peroxidation

Exposure to PTZ did not induce an increase in lipid peroxidation when compared to the animals in the control group. Furthermore, the diazepam and polydatin groups did not show any difference when compared to the PTZ group, as shown in Figure 8.

4. Discussion

Despite the wide variety of antiseizure medications available on the market, 30% of patients still suffer seizures and selecting the appropriate treatment for epilepsy remains a challenge due to the significant adverse effects associated with these drugs [30,31,32]. In this context, the search for substances that can assist in seizure control has led to numerous studies. Among the natural products investigated, polydatin stands out for its remarkable antioxidant and neuroprotective potential observed in experimental models [33,34]. Recently, Schimith et al. (2022) [18] described the properties of polydatin in various neuropathologies, indicating a lack of studies demonstrating its potential use as an anticonvulsant. Thus, this study investigated the effect of polydatin in an experimental model of convulsive-type seizures. Our study was conducted with zebrafish larvae, a species widely recognized in neuropharmacology due to its importance in drug development and research [35,36]. The use of this experimental model allows the analysis of various behavioral patterns and biochemical parameters.

In this work, we evaluated the effects of polydatin on acute seizures induced by PTZ in 7 dpf zebrafish larvae. Data related to the occurrence and latency to seizure onset in stages I, II, and III were analyzed, providing important information on the temporal dynamics of the PTZ-induced convulsive process and potential pharmacological interventions. Occurrence and latency are crucial parameters for understanding whether a seizure occurred and when it started, which can reflect the efficiency of substances in delaying or accelerating seizures [24]. When evaluating the occurrence and latency to the onset of different stages of seizures (I, II, and III), polydatin was unable to protect the animals from PTZ-induced seizures, as it did not alter the occurrence or latency in the development of seizures. However, the PTZ-treated group showed a significantly shorter latency to the progression of seizures compared to the control group. The occurrence of seizures suggests that PTZ was able to cause and reach all three seizure stages in the exposed animals. Additionally, the reduced latency in the PTZ group suggests that the convulsant accelerated seizure development. These effects are expected due to its antagonism with GABA-A receptors (gamma-aminobutyric acid type A), leading to inhibition of inhibitory neurotransmission and consequently an increase in neuronal excitability, resulting in hyperactivity in the animals [37].

The locomotor activity of zebrafish larvae is influenced by several factors, including the development of the nervous system, motor control, brain function integrity, and visual pathways [38,39,40]. A detailed analysis of spontaneous movement provides crucial information for assessing the integrity of motor systems and the efficacy of new drugs. This approach is particularly useful for identifying behavioral changes that may signal the presence of neurotoxic or neuromodulatory agents [41]. The data on spontaneous movement in zebrafish larvae were recorded in complete darkness with no stimulus to investigate the locomotor response after pretreatment with polydatin and PTZ exposure. Our results showed that polydatin was not effective in altering the behavioral changes in total distance, average speed, and mobility time of the animals caused by PTZ. However, PTZ significantly increased the analyzed parameters.

To induce acute convulsive-type seizures in zebrafish larvae, we used a concentration of 10 mM PTZ with a 60 min exposure, which has already been shown to be sufficient to induce characteristic behavioral phenotypes [42]. These findings are in line with the existing literature that describes PTZ as a well-established convulsant capable of altering locomotor activity parameters and inducing convulsive-type seizures, commonly used in experimental models of epilepsy [28,43,44,45]. Furthermore, our study using 10 mM PTZ corroborates the results of Baraban et al. (2005) [24], demonstrating an increase in seizure stages over time in zebrafish larvae.

Diazepam, a widely used benzodiazepine for the treatment of acute seizures, works by potentiating the activity of GABA, a crucial inhibitory neurotransmitter in the central nervous system [46]. Our positive control, diazepam, prevented the occurrence of seizures, increased the latency to seizure stages, and reduced behavioral parameters compared to the PTZ-treated group. These results corroborate the literature that uses diazepam as a positive control and documents its effect as an anticonvulsant drug [47].

Data from acridine orange staining provided valuable information on cell death in different experimental groups. PTZ exposure led to an increase in the number of pyknotic nuclei compared to the control group, suggesting the occurrence of cell death, a phenomenon commonly associated with pathological processes during seizures. This result is consistent with previous studies, such as Dang et al. (2021) [48], who observed apoptosis in zebrafish larvae exposed to PTZ compared to the control group, which caused neuronal damage in the animals. Our results showed that polydatin was not effective in minimizing the pyknotic points caused by PTZ. Diazepam was effective in reducing cell damage, highlighting its potential as a neuroprotective agent in convulsive seizure models.

The evaluation of oxidative stress indicated that PTZ administration resulted in a substantial increase in RS production compared to the control group. According to Jin et al. (2018) [37], PTZ induces RS production, corroborating our results. The highest concentration of polydatin, as well as the DMSO + PTZ group, showed no significant differences. Additionally, polydatin at concentrations of 200 and 300 µM showed an increase in RS production compared to the DMSO + PTZ group, which may be attributed to the effect of DMSO, present in the same concentration (0.61%). Previous studies have shown that polydatin can reduce oxidative stress and inflammation in experimental models, which could theoretically contribute to an anticonvulsant action. The literature suggests that polydatin may have neuroprotective effects, possibly modulating oxidative stress and inflammation, both of which are important factors in the pathogenesis of epileptic seizures [4,33]. Polydatin can modulate oxidative stress by increasing the activity of enzymatic and non-enzymatic antioxidants, reducing RS levels, and protecting cells from oxidative damage. Additionally, polydatin has been shown to reduce inflammation by inhibiting the activation of inflammatory cells, such as microglia, and decreasing the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukins (IL-6 and IL-1β) [18]. Despite previous studies highlighting the antioxidant effects of polydatin, in this study, polydatin was unable to prevent the increase in RS production induced by PTZ. The observed antioxidant effect may have been caused by DMSO, as this solvent was present in the highest concentration of polydatin. Several studies demonstrate that DMSO can exert potential antioxidant effects. Bulama et al. (2022) [49] showed a potential antioxidant effect of DMSO through its ability to increase the level of antioxidant enzymes that inhibit RS formation, thus improving cognitive functions. Additionally, in the study by Sanmartín-Suárez et al. (2011) [50], DMSO was reported to be effective in reducing hydroxyl radical production during auto-oxidation of compounds such as 6-hydroxydopamine. This antioxidant effect can be attributed to DMSO’s ability to act as a reducing agent, stabilizing free radicals and preventing reactions that generate more reactive species.

Data regarding TBARS levels, which are indicators of lipid peroxidation, did not show significant changes between the groups, suggesting that despite PTZ exposure, the experimental conditions did not result in perceptible alterations in this parameter in zebrafish larvae. Although the current literature does not include studies specifically on lipid peroxidation in zebrafish larvae exposed to PTZ, our data indicate that PTZ did not promote an increase in lipid peroxidation in the larvae. The results suggest that although PTZ induces neuronal stress and cell death, lipid peroxidation processes were not significantly affected under the experimental conditions used.

The authors should discuss the results and how they can be interpreted from the perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.

5. Conclusions

Although previous studies with polydatin showed promising results, they did not demonstrate efficacy in the seizure-like model used in this research. It is important to consider the differences between the experimental models employed in previous investigations and the specific model adopted in this study. A relevant factor is the complexity of the cellular and molecular mechanisms involved in epileptic seizures, which may not be fully modulated by polydatin, especially if the compound does not act on the specific targets related to the control of neuronal excitability. Furthermore, the dosage, regimen, and timing of polydatin administration may not have been optimal for seizure control, potentially compromising its efficacy. Another aspect to consider is the possibility of testing different seizure-like models. Thus, the absence of positive results in this model may indicate the need for methodological adjustments or the conduct of more in-depth studies on the mechanisms of action of polydatin in the context of seizure-like events. Despite the negative results, these findings contribute to the growing body of literature on polyphenols and emphasize the importance of standardizing experimental models and developing appropriate methods to evaluate the efficacy of neuroactive compounds.