Positive effects of angiotensin-converting enzyme (ACE) inhibitor, captopril, on pentylenetetrazole-induced epileptic seizures in mice

Purpose: To evaluate the effects of an angiotensin-converting enzyme (ACE) inhibitor, captopril, on pentylenetetrazole (PTZ)-induced seizures and post-seizure hippocampal injury. Materials: Thirty-five male Balb-c mice weighing 30 33 g were divided into control, saline PTZ, s(erum physiologic 1 ml/kg as solvent), positive control (valproic acid 200 mg/kg), captopril (25 mg/kg/day for 7 days), and captopril (50 mg/kg/day for 7 days) groups. PTZ (60 mg/kg) was administered thirty minutes after medication administration to induce epileptic seizures. The animals were observed for 30 min to record Racine stages, the time of the first myoclonic jerk (FMJ), and the occurrence of the first generalized tonic-clonic seizure (GTCS). Cornu Ammonis (CA)1, CA2, CA3, and the dentate gyrus (DG) of the hippocampus underwent histopathological examinations. The levels of total oxidant status (TOS), oxidative stress markers (total antioxidant status, TAS), and oxidative stress index (OSI) were measured in the brain tissue. Results: Compared to PTZ group, 25 mg/kg captopril decreased seizure scores and delayed FMJ and GTCS (p < 0.05). Histopathological assessment demonstrated that both 25 and 50 mg/kg captopril alleviated neuronal injury in CA1, CA2, CA3, and DG compared to PTZ (p < 0.05). Also, TOS and OSI levels in the brain tissue were reduced by both 25 and 50 mg/kg doses of captopril (p < 0.05). Conclusion: Captopril favorably improves epileptic seizure parameters and acts against post-seizure neuronal injury in the hippocampus. Captopril may be a drug of choice in epileptic individuals with hypertension.


INTRODUCTION
Epilepsy is a disorder of the central nervous system and it is characterized by a chronic and progressive nature. In epileptic patients, chronic impairments occur in the nervous system, resulting from episodes of sudden cerebral neuronal discharge. Epilepsy holds the second rank among common neurological disorders. The prevalence of epilepsy is estimated to be 2% and its annual incidence is 50 people/10,0000; contributing to 1 % of the global disease burden. The overall processes in the pathogenesis of epilepsy are complex. It involves an initial silent degeneration, later progressing to neuronal injury and ending in sclerosis. Progressive neuronal sprouting and potential consequent neurogenesis may result in pathological alterations in receptor configuration and channelopathies [1].
Angiotensin-converting enzyme (ACE) is involved in the conversion of circulating angiotensin I to angiotensin II; which is a potent vasoconstrictor and one of the main actors of remodeling processes [2]. ACE inhibitors reduce blood pressure and systemic vascular resistance and improve cardiac functions [3]. Besides the presence of the renin-angiotensin system (RAS) in peripheral tissues, RAS is available in the central nervous system with all of its components including the respective precursors and enzymes [4]. In the central nervous system, angiotensin II acts on angiotensin receptors named AT1 and AT2, playing several roles in the regulation of the cardiovascular system, appetite, stress, memory, and depression [5]. Furthermore, anticonvulsant effects of angiotensin-like peptides have been shown in experimental seizure models when those molecules were applied to brain ventricles directly (i.c.v.), suggesting a potential effect of the RAS system in the control of epileptic seizures [6].
Captopril is one of the most commonly prescribed antihypertensive drugs, selectively and potently inhibiting ACE. It is a prodrug and can pass the blood-brain barrier readily. Systemic administration of captopril is associated with the inhibition of the RAS system in the brain [7]. Compiling evidence in the recent literature demonstrate that reduced central angiotensin II activity caused by ACE inhibitors may be associated with antidepressant and anxiolytic effects [8]. Moreover, it has been suggested that ACE inhibitors, including captopril, enhance learning processes in humans [9].
This study was planned to examine the effects of captopril on PTZ-induced seizures and to prove the neuroprotective effects of captopril on CNS after PTZ administration.

EXPERIMENTAL Animals
Thirty-five male adult BALB-c Albino mice with bodyweights of 30-33 g were included in the experiment (n =35). The animals included in the experiment were kept at an ambient temperature of 22 ± 3 0 C and 12 hours of light and 12 hours of dark cycles. Ad libitum access was provided to animals for water and food. It was ensured that animals included in the experiment were acclimatized to laboratory conditions before starting the tests. All experiments were carried out in a blinded setting. The tests were conducted in the hours from 09:00 to 17:00. Each experimental group consisted of 6 animals. The Animal Ethics Committee of Cumhuriyet University approved the study protocol (Approval no:139-65202830-050.04.04-181).

Drug administration
Pentylenetetrazole (PTZ), captopril, and valproic acid (VPA) (Sigma-Aldrich Co., St Louis, MO, USA) were dissolved in physiological saline. Each drug solution was prepared freshly on the days of the experiments.

Experimental protocols
The animals were assigned to five experimental groups of 7 randomly. Group 1 was defined as the control group. The mice in group 2 received saline intraperitoneally (i.p.) at a dose of 10 ml/kg. Group 3 was the positive control group and the mice in this group received a 200 mg/kg dose of VPA i.p. The mice in group 4 and group 5 received 25 mg/kg and 50 mg/kg captopril i.p. for 7 days. Pentylenetetrazole (PTZ) was given to mice to induce epileptic seizures at i.p. doses of 60 mg/kg after the administration of the last dose of the medicine. The seizure severity was scored based on Racine's Convulsion Scale (RCS). Seizure stages are defined by RCS as follows: 0 = no convulsion; 1 = twitching of vibrissae and pinnae; 2 = motor arrest with more pronounced twitching; 3 = motor arrest with generalized myoclonic jerks; 4 = tonic-clonic seizure while the animal remained on its feed; 5 = tonic-clonic seizure with loss of the righting reflex; and 6 = lethal seizure. The mice were observed for 30 minutes after PTZ injection both for behavioral scoring according to RCS and for determining the time of FMJ, which indicated the seizure onset [10]. The animals were sacrificed by decapitation after four hours. The brain tissue obtained from the animals underwent biochemical and histological assessments.

Preparation of brain tissue homogenates
After mixing the brain tissue samples of the animals with a cold phosphate-buffered saline solution, the tissue samples were homogenized using a mechanical homogenizer (Analytik Jena  speedmill plus, Jena, Germany). The homogenates were centrifuged at 10000 × g for 10 min at a temperature of 4ºC. Then, the supernatants were obtained and stored in ice until biochemical analysis. Beckman Coulter AU680 Chemistry System (Beckman Coulter, Miami, FL, USA) and commercial kits (Rel Assay Diagnostic, Gaziantep, Turkey) were used to measure the levels of TAS and TOS. A Beckman Coulter AU680 Chemistry System (Beckman Coulter, Miami, FL, USA) was used to measure protein concentrations at tissue level based on the principle of the Bradford method [11].

Measurement of total antioxidant status (TAS)
The TAS concentrations at tissue level were determined with an automated assay method that was previously developed by Erel [12]. The method is based on monitoring the reaction rate of free radicals by measuring the absorbance of colored dianisidyl radicals during free radical reactions starting with the production of hydroxyl radicals in Fenton reaction. Antioxidants in the tissue samples should suppress coloring proportionally to their concentrations [12]. The results were expressed in micromolar Trolox equivalents per milligram tissue protein (μmol Trolox Eq/mg protein).

Measurement of total oxidant status (TOS)
Tissue TOS concentrations were quantified with the automated assay method of Erel [13]. Because ferrous ion is oxidized to ferric ion when adequate quantities of oxidants are available in the medium, the method allows for quantifying TOS levels by measuring tissue levels of ferric ions with the use of xylenol orange. Hydrogen peroxide was used for the calibration of the assay [13]. The results of the assay were expressed in micromolar hydrogen peroxide equivalents per milligram tissue protein (μmol H2O2 Eq/mg protein).

Calculation of oxidative stress index (OSI)
The ratio of TOS to TAS in percentages provided OSI to determine the severity of oxidative stress [13]. OSI was calculated as the ratio of TOS to TAS, expressed as a percentage. OSI = [(TOS, μmol H2O2 Eq/mg protein)/ (TAS, μmol Trolox Eq/mg protein) × 100] .

Histopathologic examination
After the experiments, a high dose of urethane was administered to the animals. Following this procedure, the mice underwent transcardial perfusion with 100 ml of saline and, then, a 100 ml fixative solution (formaldehyde 4 % in 0.2 M buffer phosphate at pH ¼ 7.4). After the transcardial perfusion, the mice were decapitated and their brains were collected. Until the time of histological procedures, the collected brains were left in 4 % formalin for at least 72 h. For histopathological examinations, paraffin blocks of tissue samples were prepared. Then, coronal sections of 5 mm thickness were obtained serially from these paraffin blocks. Ten sections of tissue samples, containing the hippocampus of each mouse were selected by using a systematic randomization method. Then, these tissue samples were mounted on poly-L-lysine coated slides. The tissue samples were stained with Toluidine blue to visualize dark hippocampus neurons. The tissue slides were examined under a light microscope (BX51, Japan) at X40 magnification (UPlan FI lens, Japan). Digital photographs of the microscopic images of hippocampal CA1, CA2, CA3 regions and the dentate gyrus (DG) were acquired from both hemispheres..A physical dissector method was used to perform a quantitative analysis of the dark neurons.

Statistical analysis
The data are expressed as mean ± SEM. A oneway ANOVA followed by Tukey post-hoc test was used to compare the study data. P < 0.05 indicated statistical significance.

Epileptic behavior
The RCS epileptic seizure scores were statistically significantly different between the saline group and the 25 mg/kg captopril group (p<0.05). However, no statistically significant differences in the RCS scores were found between the saline group and the 50 mg/kg captopril group (p>0.05) ( Table 1). The FMJ time was statistically significantly different between the saline group (1,24 ± 0,03 min) and the 25 mg/kg captopril group (p<0.05). The difference in FMJ time between the 50 mg/kg captopril group and the saline group was not statistically significant (p>0.05) ( Table 1). The GTCS onset was statistically significantly different between the saline group and the 25 mg/kg captopril group (p<0.05) but it was not statistically significantly different between the 50 mg/kg captopril group and the saline group (p>0.05) ( Table 1).

Histopathological features
Dark neurons were identified with the presence of neuronal shrinkage, cytoplasmic eosinophilia, Data are presented as mean ± SEM; *p < 0.05 in comparison to PTZ group nuclear pyknosis, and surrounding spongiosis in CA1, CA2, CA3 regions of the hippocampus and DG ( Figure 1, Figure 2, Figure 3, and Figure 4). Both the 25 mg/kg and 50 mg/kg doses of captopril and VPA significantly prevented dark neuron formation in CA1, CA2, and CA3 regions of the hippocampus and DG after PTZ induced seizures (p< 0.05, Figure 5A, 5B, 5C, and 5D).

Biochemical characteristics
Treating the mice with VPA and 25 mg/kg and 50 mg/kg doses of captopril significantly reduced brain TOS levels compared to the mice in the PTZ group (p <0.05) ( Table 2). Furthermore; VPA and 25 mg/kg and 50 mg/kg doses of captopril decreased OSI levels in the brain compared to the PTZ group (p <0.05) ( Table 2). However, neither VPA nor both doses of captopril acted on the brain TAS levels (p>0.05, Table 2).

DISCUSSION
The current study reveals that a potent ACE inhibitor, captopril, had anticonvulsant activity against PTZ-induced seizures; it prevented dark neuron formation in the hippocampus after PTZinduced seizures, and it alleviated oxidative stress after PTZ-induced seizures in brain tissues.     Data are presented as mean ± SEM; a p < 0.05 compared to control group and p < 0.05 compared to saline (PTZ) group An earlier study has reported that captopril had an anticonvulsant effect against strychnineinduced seizures dose-dependently [14]. Strychnine is a highly selective and potent competitive antagonist of glycine. It shows convulsant activity by acting on strychninesensitive inhibitory glycine receptors in the spinal cord and the brainstem [15]. Therefore, the anticonvulsant effect of captopril can be mediated via the inhibition of spinal postsynaptic glycine [14]. Furthermore, GABAergic inhibitory neurotransmission should be considered to interpret our study results since captopril contains a GABA precursor, proline, in its structure. Moreover, PTZ is a GABAA receptor antagonist [14]. In the central nervous system, GABA acts as the major inhibitory neurotransmitter. Also, it is responsible for seizure control [16]. Interactions between angiotensin II and GABA have been suggested to play a role in seizure susceptibility. Angiotensin II potentiates the inhibition caused by GABAergic agonists. For example, GABA itself and muscimol have been demonstrated to act on the threshold of pentylenetetrazoleinduced seizures. Also, the seizure threshold was increased by the administration of bicuculline, which is a competitive antagonist of GABAA receptors [6]. Another study showed the allosteric activation of GABAA receptors after the administration of a combination of captopril and PB [17]. In contrast to these studies, captopril alone showed no effects in maximal electroshock seizure (MES) models but it increased the anticonvulsive effects of CBP and LTG [18]. These different effects have been reported by experimental studies, showing that PTZ acts on the GABA pathways and MES models act on Na + channels.
In recent years, several studies have shown that oxidative stress and inflammation took a critical part in the pathogenesis of epileptogenesis. It has been reported that these molecules reduce oxidative stress and inflammation levels in tissues and they significantly increased the epileptic threshold [19]. Several experimental studies demonstrate that captopril shows antioxidant and neuroprotective effects under different conditions in the rodent brain. A study reported that captopril improved the memory function by diminishing oxidative stress in the hippocampus [20]. Parallel to the findings of the previous study, a study by Abareshi et al has shown that captopril preserved lipopolysaccharide-induced learning and acted against memory impairments by reducing oxidative stress levels [21]. Captopril has been demonstrated to show positive effects on STZinduced dementia in the rat in association with reduced oxidative stress levels [22]. Furthermore, neuroprotective effects of captopril were shown in the dopaminergic neurons of the nigrostriatal system in animal models of Parkinsonism [23]. In the present study, we have demonstrated that captopril reduced oxidative stress parameters similar to the previous studies in the literature as discussed above. This finding can explain the anticonvulsive and neuroprotective effects of captopril on other pathways, excluding its actions on the GABAA receptor.
Dark neurons are basophilic cells that undergo morphological changes in the central nervous system. They might be detected following some pathological conditions; including hypoglycemia, ischemia, and stress in the brain [24]. Epilepsy has also been proposed as a primary reason for dark neuron production [25]. Parallel to the results reported by previous studies in the literature, the present study demonstrated dark neuron formation in the hippocampus after the development of PTZ-induced seizures in mice [26]. Several studies have also confirmed that seizures result in hippocampal injury [27]. These can explain the increased levels of oxidative stress parameters in brain tissue after PTZinduced seizures. These harmful reactive oxygen species can lead to damage in the hippocampus.

CONCLUSION
The findings of this study indicate that captopril has antiepileptic properties and protects hippocampal neurons after epileptic seizures by reducing oxidative stress levels and increasing GABA flux into neurons. This fact should be taken into consideration in the management of epileptic patients suffering from hypertension. However, further studies are warranted to elucidate the anti-epileptic effects of captopril.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that the authors named in this article conducted this study. The authors will carry all liabilities concerning rights relating to the content of this study. The paper was read thoroughly and confirmed by all authors.

Open Access
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