INTRODUCTION:

Epilepsy is a neurodegenerative disorder marked by unpredictable seizures that range from a momentary gap in attention and muscular spasms to acute and long-lasting seizures. Epilepsy affects around 65 million people globally¹. Although many epilepsy patients can be properly treated with currently available anti-epileptic (AEDs) medications, up to 30% of this patient population does not achieve appropriate seizure control².

Despite the fact that the acute pentylenetetrazol (PTZ) rodent seizure model was created over 60 years ago, it is currently utilized to evaluate prospective anticonvulsant drugs³. Action of AEDs in different ways, but the most important and common are GABA stimulators, glutamate antagonists, calcium blockers and sodium channel blockers. Epilepsy is commonly caused by intracranial injury, neoplasms, brain inflammation, and apoplexy⁴. GABA is a neurotransmitter that controlled epilepsy. Glutamate is an excitatory substance that promotes neuronal death and is linked to epilepsy's glutamate neurotoxicity⁵. Zonisamide is a new antiepileptic medication with the chemical formula 1,2- Benzisoxazole-3-methanesulfonamide. Zonisamide belongs to the methanesulfonamide family of chemicals. It has the same sulfamoyl group as acetazolamide, an arylsulfonamide analogue with anticonvulsant properties⁶. Zonisamide has an anticonvulsant effect (fig 1) similar to phenytoin, carbamazepine, and sodium valproate, in that it prevents seizure discharges from spreading or propagating⁷.

Figure 1: Mechanism of action of Zonisamide

Vitamin E (α-tocopherol) a lipid-soluble anti - oxidant that blocks the radical chain reaction from propagating in the liposome’s part of the biological membrane, has received a great deal of press. It can cross the blood–brain barrier and accumulate in the central nervous system at therapeutic levels, reducing oxidative stress markers. It was discovered that α-tocopherol reduces the severity of pentylenetetrazole-induced convulsions. In rodents, α - tocopherol was found to be very effective which prevents PTZ induced convulsions⁸.

Zebrafish Animal has a human-like genetic structure. Around 70% of human genes are shared, and Eighty four percent of human illness genes are expressed in zebrafish⁹. Zebrafish has central nervous system is similar to Human body that is tiny, low-cost, and genetically tractable¹⁰. In current history, the use of zebrafish as a alternate for rats as well as other laboratory animals in the analysis of bio-molecular mechanistically neurocognitive imbalances and the monitoring of impending therapeutic drugs have grown in popularity. In comparison to rodents, the time and expense required to conduct the experiment in zebrafish seem to be lesser. Zebrafish has a usual color preference that has attracted little attraction in rodent studies. Learning, choice, cognition, and visual intolerance may all be exaggerated by natural color preferences for a specific colour¹¹.

Pentylenetetrazole (PTZ) drug is used in zebrafish to provoke epileptic seizures and study seizure mechanisms. Gamma-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the brain, and PTZ causes seizures by inhibiting GABA activity at GABAA receptor¹². Zebrafish have a natural colour preference, but it has expected little consideration in previous studies. Natural color preference for a specific color can alter memory, learning, decision making, and visual discrimination¹³.

According to existing research, the main objective of the lessons was to create a zebrafish model of cognitive dysfunction brought on by epilepsy and to recreate the clinical situation that demonstrates how both AEDs and epilepsy have a negative impact on cognitive functioning. Studying zebrafish behaviour, neurotransmitter levels, and gene appearance are of utmost importance and crucial elements in creating an amazing animal model since epilepsy involves significant molecular and physiological factors. Zonisamide was tested on rodents and it shows anti-epileptic effect. α- tocopherol has anti-oxidant property and neuro protective activity. However, the combination effect of zonisamide and α- tocopherol against PTZ induced Zebrafish was not performed that’s why we established the combination effect.

MATERIALS AND METHODS:

Chemicals: γ-aminobutyricacid (GABA), α-tocopherol and Pentylenetetrazole(PTZ) were Purchase from Sigma–Aldrich. Zonisamide standard drug from Eisai pharmaceuticals.

Animal Care: Adult wild type (3-5cm) zebra fish (Danio rerio) of either sex will be acclimated in the experimental room for at least 14 days. The fish were kept at a emperature of 28°C, a pH of 6.8–7.1, and a light intensity of 250 lux with a 14hour light cycle and a 10-hour day cycle. Tetramin Tropical flakes were fed twice a day to the fish. Standard size Zebrafish tank (36 cm x 26cm x 22cm) with circulates water system and constant aeration. Females and Males have separate housing arrangements in group housing (10-12 fish per tank). All experimental procedures were approved by the institution Animal Ethical Committee (IAEC) of Acharya and BM Reddy College of pharmacy, Bengaluru, IAEC number: IAEC/ABMRCP/2020-21/17¹⁴.

Treatment Groups: Adult male zebraﬁsh were used. Zonisamide and Pentylenetetrazole were dissolved at separate beaker in 10% DMSO solution. Zebrafish were selected at three month old age with 0.5–0.6g of weight range. Animal were divided into different groups like, Group I: Vehicle control (10% DMSO); Group II: Pentylenetetrazole(PTZ) 170 mg/kg (PTZ- Positive control group); Group III: Zonisamide 10 mg/ 400 ml; Group IV: Zonisamide 10mg/ 400ml + α- tocopherol ( 1µg/g); Group V: Zonisamide 20mg/400ml ; Group VI: Zonisamide 20mg/ 400ml + α- tocopherol ( 1µg/g).

Acute toxicity study: For each test concentration, seven fish will be used, including one as a control. A geometric series of 5 different concentrations with a factor of preferably not more than 2.2. The test series will be supplemented by a blank control group. For at least 24, 48, 72, 96 hours, the fish will be monitored. If no movement is visible, the fish will be considered dead. OECD guideline 203 is to be followed¹⁵.

Intraperitoneal Injection: The vehicle group, PTZ, and AED groups were given intraperitoneally (through the posterior pelvic girdle into the abdominal nook) with a micro syringe 10µl. The experiment conducted in an isolated behaviour room with a specific temperature of 28°C±2°C and a humidity of 50–60%. To avoid a novel tank response, all of the fish were acclimatize 2hours before the experiment in the performance room. Small injection volumes of 10µl per gram of fish and a 35gauge needle are used as precautions. To alleviate distress, the zebrafish were immobilized in sponge-soaked water with benzocaine as an anaesthetic agent. Individually collected fish were placed in anesthetic solution (Benzocaine 30mg/L) and weighed to determine amount of dose and volume of injection. A soft sponge, about 20mm tall, was soaked in water and located in a 60mm Petri dish. To restrict and keep the zebrafish for injection, a 10-15mm deep slit was created into that sponge. The needle was implanted further into center line among the pelvic fins by means of a dissecting microscope to give an intraperitoneal injection. Amounts appropriate for the body weight were administered. The zebrafish were immediately placed in to the tank after being injected¹⁶.

PTZ Induced Model (n=6): The zebra fish were given an intraperitoneal injection of vehicle/AEDs and then acclimated for about 15minutes in the investigational tank prior to actually receiving PTZ. The control group expected 10% DMSO and was transferred for behavior analysis. The fishes were given PTZ (170mg/kg, IP) 15 minutes after receiving the vehicle/AEDs, and observed the characteristics, intensities, and latency were used to get the seizure scores. After taking PTZ seizure persist for about 10mins, as following it decreases with time. Based on the findings using a specified scoring method, adult zebrafish were evaluated in an experimental tank, and convulsions ratings were produced. Seizure grade, start, total distance travelled, and duration of time in the top and bottom sections of the tank were among the variables recorded¹⁷.

Behavioural analysis: One long (18') and two short (12') arm make up the T-maze. Among two short, one of the short arms of T-maze is linked to a larger rectangular chamber (9× 9') where the fish can rest safely. The compartment, which is wide and deep than other arms of the T-maze, is a perfect substrate for fish, and they expend the greater part of their time there once they find it. T-maze behaviour test was conduct in a behaviour room with a favorable temperature of 25 – 26°C and a humidity of 50 – 60%. Each zebrafish was placed at the start of the long arm during the 5-minute exploration period, and the duration it needs to complete the deeper chamber was recorded. Transfer latency was defined as the time it took the zebrafish to move into the depth compartment. After PTZ exposure, transfer latencies were measured. The T-maze test and behavioral characteristics in seizure activity were investigated (fig 2). The computer application was use to track the locomotors pattern¹⁸.

Figure 2: T-maze apparatus

Biochemical Analysis:

Fish were sacrificed after behavioural testing, and the brains were removed on ice, transported into microcentrifuge tubes, then kept at -80°C. Brains were mixed and homogenized in 570μL of 50mM Tris-HCl buffer, pH 7.4. The sample (fig 3) was then centrifuged further (3000rpm for 10minutes at 4°C), and the supernatants were utilized in later studies¹⁹.

Figure 3. Schematic representation of the investigational protocols for Biochemical Assay

GABA uptake:

The fish were anaesthetized and then euthanized by decapitation for total brain excision. The forebrain, midbrain, and hindbrain were dissected out into Petri dishes that were humidified with Hank’s balanced salt solution (HBSS- HEPES buffer). Each brain structure was separated and transferred to 24-well culture plates containing 0.5mL of HBSS-HEPES buffer. Only one isolated structure was transferred to each well and all plates were maintained at 37°C throughout the experiments. Total GABA uptake was measured with the addition of 0.33μCi mL−1 L-[3H] GABA to the incubation medium at 37°C by liquid scintillation. In all trials, the uptake was stopped with two subsequent washes after a period of 7min with two subsequent washes (1ml ice-cold HBSS-HEPES buffer). Na+ independent GABA uptake was measured as above, except that N-methyl-d-GABA was used instead of sodium. Na+ dependent GABA uptake was measured as the difference of incorporated radioactivity between the total GABA uptake and the Na+- independent GABA uptake²⁰.

Cortisol analysis:

Brain cortisol levels were used as an indicator of stress response. Anesthetize a fish with 0.2% Tricaine, Place the fish on a dissection mat after softly wiping it dry with a paper towel. Start by unpinning the fish and using a razor blade to remove the head. Remove as much soft tissue from the ventral side of the skull as possible with forceps. Using small spring scissors, cut off the eyes. Remove the bone from the ventral side of the brain by breaking open the skull. Remove the skin and skull bones from the dorsal side of the brain and place it in a PBS dish. Cortisol was estimated using the Cortisol ELISA kit (Enzo) as per the described procedure²¹.

Determination of antioxidant enzymes:

Superoxide dismutase (SOD) is a family of metallo enzyme that catalyses the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide. SOD activity was quantified in a micro-plate reader and expressed as unit SOD/milligram protein (Bannister et al., 2017). Catalase (CAT) activity was assessed by measuring the decrease in hydrogen peroxide absorbance at 240nm by ultraviolet spectrophotometry. The assay mixture had 1mL potassium phosphate buffer (50mM, pH 7.0), 0.05mL H2O2 (0.3M), and 0.01mL homogenate (20–30μg of protein). Results were expressed as µmoles of H202 metabolized/mg protein/min²². Lipid peroxidation was estimated by thio-barbituric acid reactive substance (TBARS) production . 0.1 ml of homogenate was mixed with 0.2ml of 0.1% sodium dodecyl sulphate, 1.5mL of20% acetic acid solution, and 1.5mL of 0.8% of Thiobarbituric acid solution. The amount of the mixture was diluted to 4mL using distilled water and heated for 1hour in a water bath at 95℃. Then after cooling at room temperature, the red pigment was extracted by the adding of n-butanol: pyridine (15:1) and mixture was vortex firmly. The organic layer (upper layer) was separated after 10 minutes of centrifugation at 4000rpm, and the absorbance measured at 532nm using spectrophotometer against reference blank. The tissue MDA level was determined from standard curve. The MDA level was calculated from standard curve and represented as nmole/mg of protein²³.

Statistical Analysis: All the values are expressed as Mean±SEM(n = 6). Data was analysed by ANOVA followed by Dunnett test. *P<0.05,** P<0.01,***P<0.005 is considered as significant on comparing treatment groups with control group for NTT, LDT test and comparing treatment groups with positive control group for ASC test. Statistical analysis was performed using Graph Pad Prism 5 (version 5.0.1).

RESULTS

Behavioural effects (PTZ Induced Model):

Figure 4: Displays The PTZ group was found to be predictable in their swimming and spent much more time in the lower half of the tank compared to the upper half, while the control fish spent the same amount of time in both halves of the tank. Drug therapy reversed the PTZ seizure effect and time spent in the lower half of the tank because fish were protected from seizures by AED treatment. Therefore, compared to the PTZ group, the AED treated group stayed longer in the upper half.

Figure 4: Pentylenetetrazole (PTZ) induced locomotor pattern and behavior. (A)(B) Represent the total time spent by each ﬁsh in the lower and upper half of the experimental tank. (C) Represent the total distance traveled by the ﬁsh in experiment tank in different groups. Data are expressed as Mean ± SEM, n = 6 and statistical analysis by one-way ANOVA followed by Dunnett test ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

T-maze test:

Figure 5: T-maze analysis and tracking pattern. (A) represent total time spent in the wrong arm of T-maze by each ﬁsh (B) represent total distance travelled to reach the deeper chamber. Data are expressed as Mean±SEM, n = 10 and statistical analysis by one-way ANOVA followed by Dunnett test ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 5: Displays The PTZ group demonstrated an opposite effect from that of the control group, which showed little to no repeated entry into the wrong arm. As a result, the PTZ group's time and overall distance travelled were found to be significantly less than those of the control group. Zonisamide and α tocopherol treated group displayed memory impairments similar to PTZ group, taking a high/long time to reach the deepest chamber and spending more time in the incorrect arm.

Cortisol estimation:

Figure.6: Effects of zonisamide and α-tocopherol in Cortisol levels. Values were expressed as Mean±SEM (n = 6). Data was analyzed by ANOVA followed by Dunnett test. *P<0.05, **P<0.01 ***P<0.001 is considered as significant on comparing treatment groups with positive control groups.

Figure 6: Displays the effects of zonisamide and α-tocopherol in cortisol level. Cortisol levels in the treated groups significantly decreased when compared to the positive control group. As a result, we can conclude that perhaps the treatment group reduces oxidative stress in the brain.

GABA uptake:

Figure 7: Effects of Zonisamide and α-tocopherol in GABA levels. Values were expressed as Mean ± SEM (n = 6). Data was analyzed by ANOVA followed by Dunnett test. *P<0.05, **P<0.01 ***P<0.001 is considered as significant on comparing treatment groups with positive control groups.

Figure 7: Displays the effects of zonisamide and α-tocopherol on PTZ induced epileptic seizure in GABA level. Neurotransmitter analysis showed that, as PTZ is a GABA-A receptor blocker the levels GABA in PTZ treated group were lower compared to control. No signiﬁcant increase in GABA was found Zonisamide and α- tocopherol treated groups when compared to the PTZ group.

Effects of zonisamide and α-tocopherol in SOD activity:

Figure 8: Effects of zonisamide and α-tocopherol in SOD levels. Values were expressed as Mean ±SEM (n = 6). Data was analyzed by ANOVA followed by Dunnett test. *P < 0.05, **P< 0.01***P<0.001 is considered as significant on comparing treatment groups with positive control groups.

Figure 8: displays the effects of zonisamide and α-tocopherol in SOD activity. When compared to the positive control group, zonisamide 10 mg/400 ml +20 mg/400 ml α-tocopherol combination showed an increase in SOD concentration, indicating that medication therapy effectively enhances the body's own primary defense system.

Effects of zonisamide and α-tocopherol in Catalase activity.

Figure 9: Effects of zonisamide and α-tocopherol in Catalase activity. Values were expressed as Mean±SEM (n = 6). Data was analyzed by ANOVA followed by Dunnett test. *P< 0.05, **P< 0.01 ***P<0.001 is considered as significant on comparing treatment groups with positive control groups.

Figure 9: displays the effects of zonisamide and α-tocopherol in Catalase activity. When compared to the positive control group, the treated group showed an increase in Catalase concentration. Zonisamide 20 mg/100 ml α-tocopherol combination shows better activity than zonisamide 10 mg/100 ml α-tocopherol combination. No significant effect was shown by Pregabalin low concentration. This indicates that medication therapy effectively enhances the body's own primary defense system.

Effects of zonisamide and α-tocopherol in MDA activity.

Figure 10: Effects of Zonisamide and α-tocopherol in MDA levels. Values were expressed as Mean±SEM (n = 6). Data was analyzed by ANOVA followed by Dunnett test. *P<0.05, **P<0.01 ***P< 0.001 is considered as significant on comparing treatment groups with positive control groups.

Figure 10: Displays the effects of zonisamide and α-tocopherol in Catalase activity. When compared to the positive control group, the treated group showed decrease in MDA concentration. No significant effect was shown by zonisamide low concentration. It suggests that medication therapy can effectively protect against tissue damage.

DISCUSSION:

Epilepsy is a neurological condition that simplifies life expectancy and is characterised by unprovoked recurrent seizures. Patients with epilepsy experience impairment, mortality, chronic conditions, marginalization, and financial burdens due to seizures. The complex mechanism of epilepsy cannot only be understood through human clinical studies, despite the importance of human neurological and genetic tests in explaining the aetiology of neural diseases²⁴^.Due to the fact that many patients have already been exposed to a variety of anti-epileptic medications, there are ethical concerns, a lack of sample numbers, and a need for proper controls in human clinical studies (AEDs) ²⁵. In spite of these drawbacks, zebrafish remain one of the most effective experimental organisms for neurological research. Their weekly egg production of hundreds, 70% human genome similarity, robust behaviour, and physiological phenomena make them ideal for mass drug screening and disease modeling²⁶.

In present study controlling seizures is possible with proper anti-seizure medication use, up to 70% of epilepsy sufferers could go seizure-free. After two years without seizures, stopping anti-seizure medication may be an option; however, pertinent clinical, social, and private considerations should be made. The two most reliable indicators of seizure recurrence are an identifiable seizure aetiology and an abnormal electroencephalography (EEG) pattern.

Although midazolam IM has become the benzodiazepine of choice for treating status epilepticus outside of the hospital and a reliable substitute inside the hospital, elevated clinical studies are still suffering from a lack beyond this early stage. However, ethosuximide and valproate have been shown to be the most effective AED in absence epilepsy, while the majority of epilepsy treatments still lack a strong evidence base. Tonic-clonic seizures in generalised epilepsy are being treated with the new medications perampanel and lacosamide^27.

Oxidative stress is mainly involved in the age progression mainly creating an imbalance in the redox state and thereby generating ROS. ROS is the main mediator for brain injury. The ROS mainly initiates the lipid peroxidation and H2O2 damages the brain. The oxidative stress mainly impairs the cognitive function, judgement, learning and memory resulting in neuronal apoptosis. We examined the neuroprotective effect Zonisamide and α-tocopherol based on the in-vivo antioxidant model^28.

In our study according to behavioural analysis and locomotor tracking patterns, combination of zonisamide high dose and α tocopherol assisted fish in overcoming the PTZ effect and returned normal swimming movements almost everywhere in the tank. zonisamide high dose and α tocopherol treated group shows they spent more time in upper half than compare to PTZ treated group. The locomotor moment of the fish was seen in the bottom-most area of the tank in the PTZ-treated group, and the fish also spent more time there, which is similar to clinical stupor-like behaviour and anxiety in epileptic condition. In the current study, all of the fish treated with PTZ had lower levels of GABA, which contribute to epileptogenesis. GABA levels were found to be high in the control group as well as zonisamide treated group that act via a GABAergic pathway. Neurotransmitter analysis showed that, as PTZ is a GABA-A receptor blocker the levels GABA in PTZ treated group were lower compared to control. No signiﬁcant increase in GABA was found Zonisamide and α- tocopherol treated groups when compared to the PTZ group.

In zebrafish, cortisol is the primary stress hormone after the activation of the HPI axis, like humans. The amount of cortisol in zebrafish increases in response to locomotor behaviors. PTZ induction increased cortisol levels in zebrafish in our study, but zonisamide low concentration as well as zonisamide high concentration + α-tocopherol combination reduces the cortisol response to seizure activity.

Biochemical parameters of various enzymes when examined showed a favorable outcome with treatment group. Zonisamide, at concentrations of 10mg/400mL and 20mg/400mL with α-tocopherol, demonstrated good antioxidant activity by increasing the levels of antioxidant enzymes in the brain. The antioxidant enzyme level increased and had a protective effect against the induction of PTZ. Enzymes like SOD and Catalase were found to be significantly abundant in the brain. MDA levels are primarily responsible for the anti-inflammatory property. When the treatment group was used, MDA levels decreased. It indicates the effectiveness of the drugs.

CONCLUSION:

In the present study, the treatment with zonisamide + α-tocopherol combination shown significant improvement in the behavioral models such as PTZ induced model, and T-maze test. The treatment with zonisamide + α-tocopherol combination increases the level of neurotransmitter in response to PTZ induction. In PTZ induced zebrafish, treatment with zonisamide+α-tocopherol combination results in a significant reduction in cortisol levels. The levels of antioxidant enzyme system improvement were proved when treated with zonisamide+α-tocopherol combination. Enzymes such as SOD and catalase showed an increased levels in the zebrafish brain. The oxidative the enzyme stress parameter MDA shown a decrease in concentration of when treated with zonisamide +α-tocopherol combination. Developing an animal model for brain disorders, particularly epilepsy, is difficult due to the disease's complexity and variation among species. However, by incorporating a variety of different techniques and parameters in the zebrafish, we can study the molecular and behavioural basis of this disease.

CONFLICT OF INTREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors are express heartiest gratitude to Dr Bela Zutshi, Profess, Bangalore University for Zebrafish authentication.

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Received on 13.07.2024 Revised on 05.02.2025

Accepted on 18.06.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3777-3784.

DOI: 10.52711/0974-360X.2025.00544

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.