1. Introduction

Epilepsy is a common neurological disorder after a stroke. The disease is characterized by dysfunction of brain neurons, which manifests in the form of periodic and unpredictable seizure [1]. Researches have shown that brain function is affected by intestinal microbiota [6]. The natural gut flora play role by altering immune responses, producing essential metabolites and neurotransmitters [9]. These changes can subsequently effect on all pathways involved in neuronal stimulation and inflammation [10]. Elimination of the intestinal natural micro flora by the use of antibiotics will remove their beneficial effects [23-25]. The aim of this study was to investigate the effect of intestinal natural micro flora removal on seizure susceptibility and seizure behavior modification with the use of probiotics in male Wistar rats.

2. Materials and Methods

This study was performed on 32 male Wistar rats with weight range 200-250 gr. The mice were prepared from the Razi Institute of Tehran. Animals were maintained under controlled temperature (22±2° C) and a 12:12-h light: dark cycle. The animals were randomly divided into four groups: (1) Control group, (2) Antibiotic group, (3) Probiotic group and (4) Antibiotic + probiotic group. To remove the micro flora, antibiotics (neomycin, ampicillin and metronidazole) for three weeks and for replacement of micro flora, probiotics (Lactobacilli casei, Lactobacillus acidophilus and Bifidobacterium bifidum) for four weeks were administered [26, 27]. Seizures were performed by intraperitoneal injection of pentylene tetrazole (45 mg/kg). The time between injection of pentylene tetrazole and the start of the second also fifth stage of seizures, the length of time the animals are in stage five seizure and Maximum seizure stage, measured as an important indicator of seizure behavior [28]. The micro flora was examined by the MRS Agar medium and the Pure Plate method [27]. The data were statistically analyzed in Graph Pad Prism v. 8.

3. Results

The results of this study showed that the use of antibiotics lead to decrease the number of intestinal bacteria (P<0.0001), increased the severity and stability of seizure stages (P<0.05) and decreased the time delay of seizure onset (P<0.05) compared to the control group. Probiotic consumption by modifying the intestinal micro flora (P<0.0001) reduced the severity of seizure and increased the time delay of seizure onset (P<0.05).

4. Discussion 

The results of the present study showed that the use of antibiotics reduces the beneficial bacteria in the gut, including lactobacillus and bifidobacteria. Similarly, other colleagues achieved these results [26, 29]. Antibiotic treatment also increased the severity of seizures and reduced the time required for seizures to occur in the seizure model caused by pentylene tetrazole. This means that removing the natural intestinal flora can subsequently eliminate the effects of beneficial bacteria. For example, Jesus-Servando et al. showed that chronic stress can facilitate epilepsy in an animal model by altering the gut microbial profile [40]. Xie et al. reported that ketogenic diets alter intestinal microbiome in resistant epileptic children [41]. 

Comparison of bacterial colony counts in rat feces showed that taking probiotic supplements, a mixture of lactobacillus and bifidobacteria, could restore the population of natural intestinal flora lost due to antibiotic treatment. Allori et al. in the animal and Plummer et al. in the human model proved that taking probiotic supplements could restore the bacteria removed from the gut [27, 31]. Comparison of seizure behavior parameters in different groups showed that the use of probiotic supplementation moderated the seizure behavior. Similar to the present study, Bagheri and her colleagues reported a reduction in pentylene tetrazole-induced Kindling attacks due to probiotic use [44]. 

5. Conclusion

It seems that elimination of micro flora has the potential to induce seizures, which can be compensated by administration of probiotics.

Ethical Considerations

Compliance with ethical guidelines

This study with Ethics Code IR.ARAKMU.REC.1395.176 was approved by the Research Ethics Committee of Arak University of Medical Sciences. 

Funding

This article was supported by Islamic Azad University of Tehran, Science and Research Branch, (Code: 9412) and exteracted from the PhD. dissertation of the first author Department of Biology, Faculty of Basic Science, Science and Research Branch, Islamic Azad University.

Authors' contributions

All authors contributed in preparing this article.

Conflicts of interest

The authors declared no conflicts of interest.

Acknowledgements

The authors would like to thank the Islamic Azad University of Tehran, Science and Research Branch for financial support.

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