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Abstract

Endothelin-1 has been shown to increase neuronal activity and glutaminergic synaptic transmission by endothelin-A receptors (ET_AR) in the nucleus tractus solitarius neurons that play an important role in epileptic seizures. Therefore, BQ-123 as an ET_AR antagonist might attenuate neuronal excitability and glutaminergic synaptic transmission. The main purpose of the present study is to investigate the protective effect of acute BQ-123 treatment against pentylenetetrazole (PTZ)-induced tonic–clonic seizures. Wistar albino rats were divided into three groups: control, PTZ, and PTZ + BQ-123 groups. BQ-123 (3 mg/kg, intravenously) was administered for 15 min before injecting with PTZ (50 mg/kg, intraperitoneally). We determined a delay resulting from BQ-123 in “duration of the seizure onset.” “Number of rats with major seizure” also decreased according to scoring with video camera in PTZ + BQ-123 group. In BQ-123-treated group, there were eight rats without a major seizure, but only one rat had a delayed major seizure. The brain tissue glutathione peroxidase activity was significantly decreased in the PTZ and PTZ + BQ-123 groups. According to the results of the control group, there was a significant increase in the protein carbonyl levels of the PTZ group and a significant increase in the nitric oxide levels of the PTZ + BQ-123 group. Histological examination showed an increase in the number of neuronal hyperchromatic nucleus especially in hippocampal gyrus dentatus region of BQ-123-treated group. We concluded that BQ-123 impeded the formation and spread of seizure to a great degree. The beneficial effects of BQ-123 were comparatively supported with biochemical parameters and histological examinations.

Keywords

Pentylenetetrazole seizure BQ-123 endothelin receptor antagonist

Introduction

Various experimental models simulate human epilepsy in addition to functioning as a system for studying epileptogenesis.^1,2 In particular, the kindling model of epilepsy has become a commonly applied method for studying seizure mechanisms. A γ-aminobutyric acid antagonist, pentylenetetrazole (PTZ), which is a circulatory and respiratory stimulant, has been reported to induce generalized tonic–clonic seizures when administered in higher doses in experimental animal models. Researchers have reported that neuronal cell death of the PTZ kindling in rats has been caused by free radical production as a result of the increased activity of the glutamatergic transmitter.^3–7 Hippocampal gyrus dentatus (GD) cell proliferation was augmented with PTZ-induced seizure in the rat brain, probably through the release of nitric oxide (NO) and glutamate.^8–10

Endothelin-1 (ET-1) is secreted as a potent vasoconstrictor agent: the majority of this agent originates in the vascular endothelial cells, thereby participating in the control of vascular tone.^11–13 ET-1, which is formed by a 21-amino acid peptide, has an affinity to endothelin-A receptors (ET_AR) and endothelin-B receptors (ET_BR). ET_ARs exist in the membrane of vascular smooth muscle cells and mediate some of the action, including vasoconstriction, with increased intracellular calcium (Ca²⁺) ion concentration, mitogenesis, and anti-apoptotic effect: this leads to increased cell proliferation and cell survival. ET_BRs are found on endothelial cell membranes and act as vasodilators via NO and prostacyclin (PGI₂) secretion, which lead to cell death and ET-1 clearance.^14,15 ET-1 cannot pass the blood–brain barrier (BBB) and is synthesized in many regions of the central nervous system (CNS), including the cerebral cortex.^16,17 BQ-123 is one of the ET_AR antagonists and the first discovered endothelin receptor (ETR) antagonist. It was found that it has antioxidant effects¹⁸ and passes through the BBB in rats.¹⁹

Shihara et al.²⁰ noted the association between augmented neuronal activity and ET-1 via ET_AR in nucleus tractus solitarius (NTS) neurons. Furthermore, they predicted that ET-1 facilitates synaptic transmission; however, the mechanism of PTZ-induced seizure at the neuronal level and neuronal activity still remains unclear.

Therefore, this study was planned to assess whether BQ-123, one of the ET_AR antagonists, affects the neuronal activity of PTZ-induced tonic–clonic seizures and the oxidant/antioxidant status in the rats’ brains.

Experimental procedures

Chemicals

The determinations made on the samples using commercial chemicals that are used in this study were supplied from Sigma-Aldrich Chemical Company (Sigma, St Louis, Missouri, USA) and Merck Limited (Merck KGaA, Darmstadt, Germany). The reagents were used according to the manufacturer’s instructions.

Animals

Male Wistar albino rats (n = 23; weighing 230–300 g) were used in the experiment and were kept in quiet rooms with 12/12 h light/dark cycle (from 8:00 a.m. to 8:00 p.m.) at room temperature and free moving. The experiments were performed in accordance with “The National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23; Revised 1996).” The experiments were approved by the local ethical committee of the Medical School of Gaziosmanpasa University (2006-DHEK-006). All experiments were carried out in the Gaziosmanpasa University laboratories.

Experimental groups

For 12 h leading up to the experiments, the animals were allowed free access to water but not food to prevent aspiration of food content. The rats were randomly assigned to 3 groups: Group 1 (control): untreated, sham-operated rats (n = 7), group 2 (PTZ): animals treated with a single 50 mg/kg intraperitoneal (i.p.) administration of PTZ (n = 7), and group 3 (PTZ + BQ-123): animals treated with single 50 mg/kg i.p. administration of PTZ and 3 mg/kg intravenous (i.v.) injection of BQ-123, given 15 min before PTZ (Sigma, USA; n = 9).

We evaluated epileptic seizure activity in terms of “duration of the seizure onset” and “number of rats with major seizure” using video camera footage taken during the experimental procedures. PTZ-induced minor attacks are characterized as isolated myoclonic jerks and clonic seizures accompanied by facial and front extremity muscle clonus. PTZ-induced major attacks following the minimal seizure are characterized by head, neck, and tail extension with the loss of the tonic flexor reflex and tonic flexion–extension following the protracted clonus.²¹ The scoring for the severity of seizures was made according to the following scale:²² 0: no changes in behavior; 1: isolated myclonic jerks; 2: only some components presented atypical minimal seizures; 3: minimal seizures; 4: major seizures without a tonic phase; and 5:completed major seizures, that is, generalized tonic–clonic seizures.

Under ether anesthesia, the animals were killed 30 min after the administration of PTZ. Their brains were removed, put on a cold surface, divided into two halves with surgical knife by dissected along the corpus callosum, and then rinsed in cold saline solution. The right brain hemispheres were used for histological examination. The left brain tissue was stored at −80°C until biochemical analyses. Homogenate, supernatant, and extracted samples²³ from the brain tissue were prepared for measurement according to the biochemical parameters and protein levels (with Lowry’s method).²⁴

NO levels

Nitrite and nitrate are both index parameters of NO. The levels of nitrite and nitrate were determined by the method depending on Griess reaction.²⁵ A standard curve was established with a set of serial dilutions of sodium nitrite, and the resulting equation was used to calculate the unknown sample concentrations according to the standard curve.

SOD activity

The activity of total superoxide dismutase (SOD; copper–zinc and manganese; EC 1.15.1.1) was determined by measuring the reduction in nitroblue tetrazolium (NBT).²⁶ The measurements were carried out during the ethanol phase of the samples after the ethanol/chloroform mixture (5/3, v/v) was added and centrifuged. The SOD activity was assessed in the ethanol phase of the extracted sample. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate.

GSH-Px activity

Glutathione peroxidase (GSH-Px) activity (EC 1.6.4.2) of brain tissue was assessed using La Paglia and Andreani’s²⁷ method. Nicotinamide adenine dinucleotide phosphate (NADPH) enzyme reaction in the mixture, which other contained NADPH, reduced GSH, sodium azide, and GSH reductase, was activated by the addition of hydrogen peroxide (H₂O₂). The alteration in absorbance at 340 nm was monitored by a spectrophotometer.

TBARS level

The brain thiobarbituric acid reactive substance (TBARS) level was evaluated by a spectrophotometric method.²⁸ The measurement of the lipid peroxidation index is based on the reaction with TBA at 90–100°C. The malondialdehyde (MDA) or MDA-like substances reacts with TBA at 90–100°C. The samples with 10% (w/v) trichloroacetic acid were mixed for the precipitation of proteins, incubated at 90–100°C and were centrifuged. An aliquot of the supernatant with an equal volume of 0.67% (w/v) TBA was reacted in a boiling water bath for 10 min. At the end of the reaction, the solution was measured using ultraviolet spectrophotometry at 532 nm. The standard graph were prepared with the help of standard solution from 1,1,3,3-tetramethoxypropane. The result was obtained in nanomoles per grams of wet tissue.

PC content

The level of PC was analyzed via the method of reaction with carbonyl groups and 2,4-dinitrophenylhydrazine.²⁹ First, the samples were precipitated with 20% trichloroacetic acid and 10 mM 2,4-dinitrophenylhydrazine. Later, 2 M hydrochloric acid was added and left at room temperature for 1 h. After 20% trichloroacetic acid was added to the tubes, centrifugation was carried out at 11,000g for 3 min. Then, the pellet was washed with ethanol/ethyl acetate (1/1) solution to remove free reagents. In the final stage, the precipitated protein was redissolved in 100 mM of sodium hydroxide solution. After centrifugation (to remove any insoluble material), spectrophotometric analysis was performed at 360 nm.

TAC and TOS analyses

Erel’s methods were used for analyzing total antioxidant capacity (TAC) and total oxidant status (TOS), which are automated and colorimetric.^30,31 TAC was determined according to the whitening of the characteristic color of a more stable 2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) radical cation by antioxidants.³² TOS was determined according to the oxidation of ferrous ions in the presence of various oxidant species in an acidic medium and the ferric ions were measured by xylenol orange. The outcomes were expressed in millimoles of Trolox equivalent per liter and micromoles of H₂O₂ per liter, respectively. The precision of this procedure is evaluated lower than 3%.³³

Histological examination

After the skulls were opened, the right brain hemispheres were immersed in 10% neutral formalin and fixed for 1 week at 4°C. After rinsing them in running tap water for 12 h, the brains were dehydrated in a series of alcohols and cleared with xylene and then embedded in paraffin. The right hemispheres were cut into serial sections of 5 μm thickness in the sagittal plane using a rotary microtome (RM 2125 RT, Leica, Nussloch, Germany). The sections were then systematically sampled, placed onto gelatinized glass slides, and stored in an incubator for 8 h at 60°C (Nuve EN 400; Ankara, Turkey). Afterward, they were stained with 0.1% cresyl fast violet.

Statistical analysis

All data were analyzed using the Statistical Package for Social Sciences (SPSS; version 15.0; SPSS Inc., Chicago, Illinois, USA) for Windows software and given as the mean ± SEM. Level of significance is regarded with the value of p < 0.05. The one-sample Kolmogorov–Smirnov test was used for analyzing the groups’ distribution. The one-way analysis of variance test was performed and post hoc multiple comparisons were carried out with Tukey’s honestly significant difference test for the normally distributed groups’ variables.

Duration of the minor seizure onset in the PTZ and PTZ + BQ-123 groups was evaluated using the Mann–Whitney U test. The number of rats with major seizure was evaluated using the χ ² test. Owing to an insufficient number of rats with major seizure, duration of major seizure onset and total duration of major seizure were not statistically evaluated.

Results

Duration of the seizure onset and number of rats with major seizure were measured in each PTZ group using video cameras. When the BQ-123 + PTZ rats were compared with the PTZ group, delay in the minor seizure onset category and reduction in the number of rats with major seizure was statistically significant (p < 0.05). While only one rat in the BQ-123 + PTZ group moved into the major seizure category, in the PTZ group, six out of the seven rats had major seizure attacks (Table 1).

Table 1.

Groups showing duration of the minor seizure onset, number of rats with major seizure, duration of major seizure onset, and total duration of major seizure.^a

Groups	Duration of minor seizure onset (s)	Number of rats with major seizure	Duration of major seizure onset/total duration of major seizure (s)
Control (n = 7)	–	–	–
PTZ (n = 7)	45 82 44 62 55 52 44 Mean ± SEM: 54.8 ± 3.4	6/7	Not into major seizure 85/12 54/119 72/215 63/55 60/115 49/23
PTZ + BQ-123 (n = 9)	110 138 85 109 171 137 210 193 379 Mean ± SEM: 170.2 ± 23.7	1/9^b	Not into major seizure Not into major seizure Not into major seizure Not into major seizure Not into major seizure 245/14 Not into major seizure Not into major seizure Not into major seizure

PTZ: pentylenetetrazole.

^aOnset of (minor) seizure after PTZ injection and number of rats with major seizure in groups was statically assessed. In PTZ + BQ-123 group, onset of seizure is significantly delayed (p < 0.05), and the number of rats with major seizure is only one rat (p < 0.05).

In terms of SOD enzyme activity, no significant changes were found, in spite of increased SOD activity in the BQ-123 + PTZ group (Table 2). According to the control group, GSH-Px activity in the PTZ (p < 0.001) and PTZ + BQ-123 (p < 0.040) groups decreased significantly (Table 2). The level of TBARS, TAC, and TOS were not statistically different (Table 2). According to the control group, there was a significant increase in the PC levels of the PTZ group (p < 0.005; Table 2) and a significant increase in the NO levels of the PTZ + BQ-123 group (p < 0.008; Table 2).

Table 2.

Effect of BQ-123 to superoxide dismutase and glutathione peroxidase enzyme activities, to the levels of nitric oxide, protein carbonyl, thiobarbituric acid reactive substance, brain tissue total antioxidant capacity and total oxidant status in pentylenetetrazole-treated rat brain.^a

Groups	SOD (U/mg protein)	GSH-Px (U/g protein)	NO (µmol/g wet tissue)	PC (nmol/mg protein)	TBARS (nmol/g wet tissue)	TAC (mmol Trolox eq/L)	TOS (µmol H₂O₂ eq/L)
Control	0.012 ± 0.001	3.650 ± 0.346	2.006 ± 0.136	2.070 ± 0.156	173.495 ± 8.957	4.988 ± 0.123	5.755 ± 0.291
PTZ	0.013 ± 0.001	1.943 ± 0.255	2.932 ± 0.356	3.176 ± 0.246	181.797 ± 8.900	4.885 ± 0.168	6.382 ± 0.558
PTZ + BQ-123	0.015 ± 0.002	2.658 ± 0.217	3.426 ± 0.333	2.541 ± 0.210	160.498 ± 10.564	4.883 ± 0.250	5.963 ± 0.642

SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; NO: nitric oxide; PC; protein carbonyl; TBARS: thiobarbituric acid reactive substance; TAC: total antioxidant capacity; TOS: total oxidant status; PTZ: pentylenetetrazole; H₂O₂: hydrogen peroxide.

^aGSH-Px activity, according to the control group, in PTZ (p < 0.001) and PTZ + BQ-123 (p < 0.040) groups revealed a significant decrease. In the NO levels, a significant increase in the PTZ + BQ-123 groups was identified (p < 0.008) according to control group. According to the control group, in the PC levels a significant increase in PTZ group (p < 0.005) was identified. There was no significance between the groups in the activity of SOD in the level of TBARS, TAC, and TOS.

Hippocampus (4×), hippocampal cornu ammonis (CA; 40×), and hippocampal GD cells were stained with 0.1% cresyl fast violet for each groups. Figure 1 shows the representative photomicrographs of 5 µm thin sagittal sections. According to control groups, more increased amount of neuronal hyperchromatic nucleus in the GD region of BQ-123 group have been determined in comparison with the PTZ group. BQ-123 treatment has moderately positive effect in the short time.

Figure 1.

Representative photomicrographs of 5 µm thin sagittal section with cresyl fast violet. Hippocampal histological sections of (A) control, (A′) PTZ, and (A′′) PTZ + BQ-123 groups in rats are shown with 4× magnification. GDhistological sections of (B) control, (B′) PTZ, and (B′′) PTZ + BQ-123 groups in rats are shown with 40× magnification. CA histological sections of (C) control, (C′) PTZ, and (C′′) PTZ + BQ-123 groups in rats are shown with 40× magnification. The arrowheads indicate increased amount of neuronal hyperchromatic nucleus. CA: cornu ammonis, GD: gyrus dentatus.

Discussion

Rat seizure models have notably supplied our fundamental understanding of epilepsy. Exposure to a widespread convulsant agent (PTZ) provokes tonic–clonic seizures as well as dose-dependent and stereotyped behavioral changes. Many researchers have investigated how to prevent PTZ-induced seizures.^34–36 For instance, Gupta et al.³⁶ reported that Centella asiatica as an adjuvant precluded cognitive impairment and diminished the oxidative stress generated by PTZ kindling. Ates et al. investigated the effects of microinjecting 2-chloroadenosine (an adenosine receptor agonist) into the thalamus alone and with theophylline pretreatment on PTZ-induced tonic–clonic seizures.³⁷ They found that 2-chloroadenosine as an adenosine agonist provokes attenuated PTZ-kindled tonic–clonic seizures in Wistar rats. Moreover, they showed that the effects of adenosine analog 2-chloroadenosine were reversed by adenosine antagonist theophylline. Intrathalamic microinjection of 2-chloroadenosine and theophylline 24 h before and 1 h before PTZ treatment were evaluated in relation to seizure duration, seizure score, and cortical electroencephalography.

However, the mechanism of the epileptogenic action of PTZ is still uncertain at the neuronal level. The existence of a relationship between ET-1 and seizures has reported in the literature, but its mechanism has not been exactly understood. Furthermore, the effect of an ETR antagonist in PTZ-induced seizures has not been investigated in the reports presently available.

It is important to understand why ET-1 causes increased neuronal activity following glutamate release in a developing seizure. Shihara et al.²⁰ indicates that ET-1 increases the neuronal activity in the NTS and contributes to the control of the basal spontaneous neuronal activity of the NTS neurons via ET_AR. Additionally, they found that ET-1 augmented the responses in neuronal activity evoked by glutamate via ET_AR because it was attenuated by BQ-123; this was not true for BQ-788, an ET_BR antagonist. Thus, ET-1 may facilitate NTS synaptic transmission via ET_AR. Nagasaka et al.¹⁷ wrote that ET_AR associated with the glutamatergic system kindled convulsive behaviors, particularly barrel rolling. Therefore, we used an ET_AR antagonist, BQ-123, and we have shown that BQ-123 prevents seizures to a great extent. We clearly detected this effect during the experiment, in the video footage, and again in the scoring.

The ET_BR agonist supply has a neuroprotective effect on cerebral ischemia.³⁸ The blocked ET_ARs in our study showed the effect of ET-1 in the cells via ET_BRs. Researchers from various studies found that ET-1 leads to the release of NO and PGI₂ secretions via activation of the ET_BR.^39,40 Therefore, we expected an increase in NO levels via increased ET_BR activity from an ET_AR inhibitor, BQ-123. It was informed that ET-1 increases Ca²⁺ levels that induce glutamate release which result in increase of neuronal activity.²⁰ Our findings suggest that ET_AR antagonist increases the level of NO. Thus, BQ-123 might prevent the epileptic activity via increasing the level of NO and decreasing the Ca²⁺ and glutamate levels.

An oxidant–antioxidant imbalance in the brain is one of the most significant mechanisms for the generation of epilepsy; therefore, researchers have conducted a large number of studies on this topic regarding both the PTZ model as well as the other models.^41,42 In our present study, BQ-123 prevented the formation and spread of seizures in the epilepsy score. However, despite the positive effects of BQ-123 in the biochemical parameters, the PTZ effects were not exactly changed. This may be related to the dose of BQ-123 and the time it was given. Although some of the agents increase the TBARS level of brain tissue,⁴³ high lipid peroxidation in the PTZ group was not significant in comparison with the control group in our study. Similarly, an increased TOS level was found in the PTZ group, but it was not significant. Although SOD enzyme activity increased with BQ-123, it was not statistically significant. On the other hand, GSH-Px activity significantly decreased in the PTZ and PTZ + BQ-123 groups. If there is an increased reactive oxygen species (ROS) production, the antioxidant systems try to scavenge them to prevent tissue injury. PTZ induced high ROS production, and as a result, we measured high protein oxidation in this group. It is known that the GSH-Px detoxifies the highly produced ROS in the brain. Thus, GSH-Px might prevent the lipid peroxidation and protein oxidation. Although we did not find a significant difference between lipid peroxidation, we found an increase in PC content of PTZ group. The protein oxidation is the result of oxidative stress. The GSH-Px activity decreased in PTZ group. The balance between oxidant and antioxidant system is important to protect the tissue against the injury. The increasing oxidative damage in the PTZ-induced seizures might cause the exerting GSH-P_X enzyme. BQ-123 caused an increase in the GSH-P_X enzyme activity, but it was statistically insignificant.

Protein oxidation is a specific marker for oxidative stress and tissue function. The fact that PTZ treatment caused high protein oxidation via increased PC levels in the rats’ brain tissue means that there might be an oxidative injury in the brain. The increase in PC in the PTZ group decreased with the ET_AR antagonist BQ-123. The PC content of the brain tissue was similar in the BQ-123-treated group and the control group. This shows that BQ-123 prevents protein oxidation generated by PTZ. The BQ-123 treatment considerably reversed the oxidative effects of PTZ in brain tissue. Our previous findings indicate that BQ-123 acts as an antioxidant agent.¹⁸

Bikjdaouene et al.⁴⁴ found that melatonin has a significant anticonvulsant effect and reduces the mortality rate in PTZ-induced seizures. Other researchers assert that caffeic acid phenethyl ester (CAPE) has a neuroprotective effect on PTZ-induced seizures in mice.⁴⁵ Although there is an increased generation of ROS in mice brains when paired with PTZ, and the neuroprotective effect of CAPE is evaluated using biochemical parameters, the researchers did not evaluate the acute effects of the agent as we do in this study. Our findings may reveal notable protective effect against epilepsy because of the acute results of the ET_AR antagonist BQ-123.

The increased GD cell proliferation with the PTZ treatment in rats could be due to an excessive release of glutamate, an NO-dependent mechanism.^8,9,46 We evaluated the acute effects of BQ-123. Nevertheless, the histological assessment of the CA and GD regions in the hippocampus partially supports the acute findings. We observed an increase in the number of neuronal hyperchromatic nucleus especially in GD region of BQ-123 treatment group. It has been expected that such an increase in neuronal cell number could be due to hippocampal neuronal plasticity and neurogenesis.

In conclusion, this is the first time an ETR antagonist was used in a model of experimental epilepsy, and it has been observed to be considerably effective. In a short time of period, biochemical and some histological findings have supported the beneficial acute effects by BQ-123. We determined, for the first time, that acute BQ-123 treatment as an ET_AR antagonist impeded the formation and spread of seizure to a great degree. The following mechanism for the occurrence of its effect might be related with increased level of NO and decreased level of Ca²⁺ and then it lessens glutamate levels. Because of its effective inhibition in the formation of seizure, ET_AR antagonists might be used as antiepileptic agent.

Even though the mechanism of epileptic seizure onset still remains unclear, our results have shown that ET-1 and ET-1 receptors are included in the physiological mechanism of seizure attacks. The underlying useful effects of the ET_AR antagonist BQ-123 require further investigation in order to be sufficiently understand the context of cell mechanisms.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This research was supported and approved by The Scientific Research Fund of Gaziosmanpasa University, Tokat, Turkey.

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