@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2018;24(2):79-87

Background
Epilepsy is a known brain disorder characterized by frequent and unplanned interruptions of normal brain function, called epileptic seizures [1].
Previous Studies
... [2, 3]. Glutamate and γ-aminobutyric acid (GABA) are two neurotransmitters that have been studied extensively in relation to seizure induced by chemical kindling. Both of these systems play an important role in the epilepsy and the resulted seizure [4]. The inhibition of GABA is an important factor in the pathogenesis of anxiety [5, 6]. Pentylenetetrazol (PTZ) can induce a seizure by blocking GABA receptors and it also induces anxiety symptoms by inducing a limbic epilepsy model [7]. .... [8-15]. Lithium not only acts as a moderator, but also has anti-depressant and anti-anxiety effects [17]. ... [18-20].
Aim(s)
This study evaluated the anxiolytic effect of administration of lithium chloride (LiCl) in penthylentetrazole (PTZ)-induced seizure in rat model.
Research type
The present research was an experimental study.
Research society, place and time
This experimental study was performed on 50 male Sprague Dawley male rats between June and September in 2016.
Sampling method and number
This experimental study was performed on 50 male Sprague Dawley male rats weighted 200±20 gr.
Used devices&materials
During the study period, the animals were kept at standard temperature (25±2°C) and relative humidity (50±5%) and the 12-h light/darkness (6 am-6 pm). All experiments were performed according to the Standards for Laboratory Animal and supervised by the Ethics Committee of Islamic Azad University, Shiraz Branch (Ethics code: 95-2034-8324). Animals were randomly divided into 5 groups (10 rats per group), including control, Sham (PTZ+Saline), three experimental groups (PTZ+Li20, PTZ+Li40 and PTZ+Li80). Control group did not receive any treatment. The PTZ+Saline group received normal saline as a lithium solvent for 14 days following the five stages of PTZ-induced seizure. This group was used to evaluate the effects of PTZ on anxiety level. Three treatment groups received lithium chloride (Sigma-Aldrich, USA) by daily intraperitoneal injection (20, 40 and 80 mg/kg) for 14 days. PTZ (Sigma-Aldrich, USA) was used to induce seizure. PTZ (40 mg/kg) was injected intraperitoneally for 5 consecutive days (animals were weighed before each injection). Thirty minutes after each PTZ injection, the rats were examined for seizure. Seizures were examined using the Racine's scale [21-23]. Behavioral experiments The elevated plus maze test It is used to measure anxiety-like behaviors in rodents [24]. Open field test It is used to measure exploratory, search and anxiety behaviors [25]. Measurement of hippocampal BDNF levels After behavioral tests, animals with a lethal dose of chloroform were anesthetized. Immediately the animal head was separated and the brain completely removed from the skull and placed on the ice. Rats’ hippocampus was carefully removed from the rest of the brain under a stereoscope. After washing with saline and Tris buffer (Sigma-Aldrich, Germany), it was homogenized at 5,000 rpm for 5 min using Homogenizer (IKA, Germany). The homogenized solution was centrifuged by a refrigerated centrifuge machine (Hermle, Germany). To prevent degradation of enzymes and proteins, all experiments were carried out at 4 C (refrigerated centrifuge) and Phenylmethylsulfonyl fluoride (0.5 μm) was used as a protease inhibitor (Sigma-Aldrich, Germany) [26]. One-way analysis of variance and Tukey's post hoc tests by SPSS 22 were used to determine significant differences in the anxiety and behavioral indices among the groups.
Findings by Text
The elevated plus maze test The mean spent time in the closed arm in the PTZ+Saline group showed a significant difference with the control group (p<0.0001; Figure 1). On the other hand, there was a significant difference in three groups treated with lithium chloride (p<0.001 for all three groups). Comparison of the lithium chloride treated groups with PTZ+Saline group showed a significant difference between PTZ+Li20 and PTZ+Saline group (Figure 1, p<0.05). No significant difference was observed between the other treatment groups with lithium chloride and PTZ+Saline group. These results showed that lithium treatment in PTZ groups only at a minimum dose (PTZ+Li20) has could reduce anxiety levels. Open field test Number of squares crossed: The results showed a significant difference between the control group and the PTZ+Saline group (p<0.0001; Table 1). This difference indicated an increase in anxiety in the PTZ group compared with the control group. Also, there was a significant difference between the control group and lithium chloride treated groups (40 and 80 mg) (p<0.05 and p<0.001, respectively; Table 1). On the other hand, the results showed a significant difference between PTZ+Saline and PTZ+Li20 groups (p<0.0001). Also, there was a significant difference between PTZ+Li40 and PTZ+Li80 groups with PTZ+Li20 group (p<0.001; Table 1). In fact, the results showed that treatment with lithium at a minimum dose reduced anxiety levels following PTZ-induced seizure. Number of central squares entries: The mean number of central squares entries in the open field test showed a significant difference between the control and PTZ+Saline groups (p<0.0001; Table 1). This difference indicates a decrease in the exploratory behaviors and an increase in anxiety levels in the PTZ groups compared with the control group. This study also showed a significant difference between the control and the PTZ+Li80 groups (p<0.01; Table 1). On the other hand, there was a significant difference between PTZ+Saline group and lithium treated groups (PTZ+Li20, PTZ+Li40 and PTZ+Li80) (p<0.0001, p<0.001 and p<0.05, respectively; Figure 3). This difference indicates increased exploratory behaviors in lithium chloride treated groups, especially at the minimum dose in comparison to the PTZ + Saline group. Climbing on the wall: The results showed a significant difference between the control and the PTZ+Saline groups (p<0.001). Also, there was a significant difference between the control group and the PTZ+Li40 and PTZ+Li80 groups (p<0.001; Table 1). The results of this study showed a significant difference between PTZ+Saline group and PTZ+Li20 group (p<0.001). Moreover, there was a significant difference between PTZ+Li40 and PTZ+Li80 groups with PTZ+Li20 group (p<0.001; Table 1). Exploratory behaviors in the PTZ treated group reduced and treatment with 20 mg/kg of body weight of rats significantly increased exploratory behaviors. Number of fecal boli: The number of fecal boli left was used as an index for anxiety between the control and PTZ+Saline groups and the results of one-way ANOVA showed a significant difference between two groups (p<0.0001; Figure 5), which indicates an increased level of anxiety in the PTZ rats compared with the control group. Also, there was a significant difference between control and PTZ+Li40 and PTZ+Li80 groups (p<0.01). In addition, a significant difference was found between PTZ+Saline and PTZ+Li20 and PTZ+Li40 groups (p<0.0001 and p<0.001, respectively; Table 1). It can be concluded that treatment with lithium chloride could reduce the level of anxiety. On the other hand, there was a significant difference between PTZ+Li40 and PTZ+Li80 groups with PTZ+Li20 group (p<0.01; Table 1). Measurement of hippocampal BDNF levels PTZ-induced seizures significantly reduced the hippocampal level of BDNF in comparison to the control group (p<0.0001; Figure 2). Meanwhile, lithium chloride treatment significantly increased the level of BDNF in the experimental groups compared with the PTZ+Saline group.
Main comparison to the similar studies
... [27-40]. PTZ causes anxiety disorder in the animal model of seizure, and treatment with lithium chloride can reduce the level of anxiety in this model. Other researchers also found that PTZ-induced Kindling seizures cause anxiety symptoms [41-42]. Also, the anti-anxiety effect of lithium has been observed in previous studies [16, 17]. The present study showed that, following PTZ-induced seizures, the level of BDNF in the rat hippocampus was decreased, which can cause behavioral disorders, including anxiety. On the other hand, administration of lithium chloride could significantly increase BDNF levels in treated groups, which indicates the beneficial effects of this element. Lithium administration increases the expression of BDNF in rodent brain, especially in the limbicular and frontal cortex. Studies have also shown that for neurotrophic effects of lithium in cortical neurons BDNF expression is needed [43]. ... [44-46].
Suggestions
It is suggested that the expression of BDNF receptors and depression-like behaviors be investigated in this model.
Limitations

Conclusions
Anxiolytic-like effects of lithium may improve the symptoms of anxiety in PTZ-induced seizure.
Acknowledgments
The efforts of Research Director of Islamic Azad University of Shiraz for the essential facilities to perform this study are appreciated.
Conflict of interest
None declared.
Ethical Permissions
None declared.
Funding Sources
This study was partially supported by the research deputy of Islamic Azad University, Shiraz branch as a research project.

TABLES and CHARTS

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