Homocysteine thiolactone-induced seizures in adult rats are aggravated by inhibition of inducible nitric oxide synthase

Abstract

Homocysteine and its metabolites (homocysteine thiolactone (HT)) induce seizures via different but still not well-known mechanisms. The role of nitric oxide (NO) in epileptogenesis is highly contradictory and depends on, among other factors, the source of NO production. The aim of the present study was to examine the effects of aminoguanidine, selective inhibitor of inducible NO synthase (iNOS), on HT-induced seizures. Aminoguanidine (50, 75, and 100 mg/kg, intraperitoneally (i.p.)) was injected to rats 30 min prior to inducing HT (5.5 mmol/kg, i.p.). Seizure behavior was assessed by seizure incidence, latency time to first seizure onset, number of seizure episodes, and their severity during observational period of 90 min. Number and duration of spike and wave discharges (SWDs) were determined in electroencephalogram (EEG). Seizure latency time was significantly shortened, while seizure incidence, number, and duration of HT-induced SWD in EEG significantly increased in rats receiving aminoguanidine 100 mg/kg before subconvulsive dose of HT. Aminoguanidine in a dose-dependent manner also significantly increased the number of seizure episodes induced by HT and their severity. It could be concluded that iNOS inhibitor (aminoguanidine) markedly aggravates behavioral and EEG manifestations of HT-induced seizures in rats, showing functional involvement of iNOS in homocysteine convulsive mechanisms.

Keywords

iNOS inhibition aminoguanidine homocysteine seizures EEG rats

Introduction

Epilepsy is characterized by sudden occurrence of seizures in behavior and spiking activity in electroencephalogram (EEG). It is one of the leading neurological disorders, affecting 1–2% of the world population.¹ Despite significant improvements in epilepsy treatment, around 30% of patients are refractory to available antiepileptic drugs.² The process of epileptogenesis, regardless of the type of epileptic manifestation, is a result of imbalance between excitatory and inhibitory phenomena within the central nervous system (CNS).³ Homocysteine, thiol-containing amino acid, together with its reactive derivate homocysteine thiolactone (HT), is considered to be one of the most potent excitatory agents in the CNS.^4,5 Their elevated plasma levels have been identified as risk factors for the development of numerous cardiovascular and brain disorders, including epilepsy.^6,7 On the other hand, plasma homocysteine level is increased by classical antiepileptic drugs.⁸ Recently, inflammation has been identified as one of the important contributing factors for the development of epileptic activity.⁹

Stanojlović et al.¹⁰ showed that acute administration of HT to adult rats resulted in epileptogenic activity on the EEG with characteristic spike and wave discharges (SWDs) and convulsive episodes in animal behavior, establishing in that way a suitable model of generalized epilepsy for studying HT mechanisms of epileptic activity.

The role of nitric oxide (NO) in epileptogenesis has been highly contradictory, since there are evidences for its anticonvulsive as well as proconvulsive properties, depending on, among other factors, the source of NO production.^11,12 Constitutive isoforms of NO synthase (NOS) are responsible for the synthesis of physiologically vital amounts of NO,¹³ while inducible NOS (iNOS) produces high amounts of NO that lasts hours or days.¹⁴ iNOS has been found to be a major contributor to initiation/exacerbation of the CNS inflammatory/degenerative conditions through the production of excessive NO.¹⁵ Moreover, iNOS is found to be overexpressed in brains of humans with epilepsy, as well as in some spontaneously epileptic animals.^16,17 In a model of HT-induced seizures, the involvement of iNOS has not been studied, although anticonvulsive role of NO has been shown.¹⁸ Therefore, the aim of the current study was to investigate the effects of aminoguanidine, selective inhibitor of iNOS,¹⁹ on behavioral and EEG manifestations of HT-induced seizures.

Materials and methods

Animals

Adult Wistar rat males (180–220 g) were used in the study. The animals were purchased from local certified supplier (The Military Medical Breeding Laboratories, Belgrade, Serbia) and kept under controlled laboratory conditions (ambient temperature 21–23°C, 55–65% humidity, 12/12 light–dark cycle, with lights at 8 a.m.) for 1 week before the start of experiments. Free access to standard laboratory chow and tap water during all experimental phases was allowed to all animals.

All experimental procedures were in full compliance with Directive of the European Parliament and of the Council (2010/63/EU) and approved by The Ethical Committee of the University of Belgrade (Permission No 298/5-2).

Surgical electrode implantation

Anesthetized animals (pentobarbital sodium, 50 mg/kg, intraperitoneally (i.p.)) were placed in stereotaxic apparatus (David Kopf, Tujunga, CA, USA) and three gold-plated electrodes were implanted over frontal (2 mm rostrally to bregma and 2 mm from the median line), parietal (2 mm rostrally to lambda and 2 mm laterally to median line), and occipital (2 mm caudally to lambda) cortices for further EEG recordings.

Experimental groups

Animals were randomly assigned to the following groups: (1) control (C, 0.9% sodium chloride, n = 6), (2) aminoguanidine (AG) (100 mg/kg, AG₁₀₀ group, n = 6), (3) HT group (d,l-homocysteine thiolactone 5.5 mmol/kg, n = 9), and (4) aminoguanidine (50, 75, and 100 mg/kg) 30 min prior to HT (AG₅₀HT, AG₇₅HT, and AG₁₀₀HT groups, respectively, n = 8 per group). Drugs were freshly dissolved in saline and after adjusting the pH to 7.0 were administered in a volume of 0.1 ml/100 g rat body weight i.p.

Behavioral recordings

Convulsive behavior was assessed during 90 min after HT administration by the incidence of motor seizures, seizure latency (time to the first motor seizure sign), number of seizure episodes per rat, and their severity. Intensity of seizure episodes was determined by a modified descriptive-rating scale as reported¹⁰ previously with grades defined as follow: (1) head nodding, lower jaw twitching, (2) myoclonic body jerks (hot plate reaction), bilateral forelimb clonus with full rearing (Kangaroo position), (3) progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tail, and (4) prolonged severe tonic–clonic convulsions lasting over 20s (status epilepticus) or frequent repeated episodes of clonic convulsions for an extended period of time (over 5 min). Additionally, lethality was recorded at 90 min and 24 h after the HT injection.

EEG registration and analysis

For EEG registration, an 8-channel apparatus (RIZ, Zagreb, Croatia) was used with digital signal acquisition achieved by SCB-68 data acquisition card (National Instruments Co, Austin, Texas, USA). Sampling frequency was 512 Hz/channel. The cutoff frequencies for the EEG recordings were set at 0.3 (high-pass) and 100 Hz (low-pass) filters, while ambient noise was eliminated using a 50-Hz-notch filter. Data acquisition and signal processing were performed using LabVIEW platform software developed in the laboratory (NeuroSciLaBG, Belgrade, Serbia). The power spectra density (obtained by the method of Fast Fourier transformation, Hanning window) of the 12s epochs was plotted, and the integrated energy signals expressed as microvolts squared in hertz.

SWDs were determined as reported earlier¹⁰ using the following criteria: (1) spontaneous and generalized, rhythmic 5–7 Hz discharges, (2) with typical spike–wave complex lasting more than 1s, and (3) amplitude of at least twice the background EEG activity. The number and duration of SWD were calculated during a 90-min period after the HT administration. All SWDs were detected visually.

Drugs

All drugs used in the study were produced by Sigma Aldrich Chemical Co, St Louis, MO, USA.

Data analysis

Incidence of seizures and lethality data were evaluated by Fisher’s exact probability test. Since the normal distribution of the data on seizure latency, number, and severity of seizure episodes, as well as number and duration of SWD have not been estimated by Kolmogorov–Smirnov test, the nonparametric analyses (Kruskal–Wallis analysis of variance and Mann–Whitney U test) were used to analyze these data (*p < 0.05 and **p < 0.01). Medians with 25th and 75th percentiles were used to present the results.

Results

Behavioral effects

All animals receiving saline (group C) or aminoguanidine only (group AG₁₀₀) showed normal gross behavioral activity.

Low seizure incidence (33.3%), late seizure onset (90 (48–90) min), and low number of seizure episodes per rat (0 (0–2)) were observed upon administration of HT in subconvulsive dose (5.5 mmol/kg, i.p., group HT). Maximal severity of these seizures was of grade 2 (Figure 1 and 2(a) to (c)).

Figure 1.

The influence of aminoguanidine, selective iNOS inhibitor, on the incidence of seizures in experimental groups. Seizures were induced by HT 5.5 mmol/kg (HT, n = 9) in adult male Wistar rats. Aminoguanidine (50, 75, and 100 mg/kg) were administered 30 min prior to HT5.5 (AG₅₀HT, AG₇₅HT, and AG₁₀₀HT, respectively, n = 8 per group). Seizure incidence was calculated as percent of convulsing animals in group. The significance of the differences between the groups was estimated by Fisher’s exact probability test (**p < 0.01 vs. HT). AG: aminoguanidine; iNOS: inducible nitric oxide synthase; HT: homocysteine thiolactone.

Figure 2.

The effects of aminoguanidine treatment on median latency to the first seizure episode (a), a number of seizure episodes per rat (b), and their severity (c). Severity of seizure episodes was determined by a descriptive-rating scale with grades defined as follow: (1) head nodding, lower jaw twitching, (2) myoclonic body jerks (hot plate reaction), bilateral forelimb clonus with full rearing (Kangaroo position), (3) progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tail, and (4) prolonged severe tonic–clonic convulsions lasting over 20s (status epilepticus) or frequent repeated episodes of clonic convulsions for an extended period of time (over 5 min). The significance of the differences between the groups was estimated by Kruskal–Wallis ANOVA and Mann–Whitney U test (*p < 0.05, **p < 0.01 vs. HT and ##p < 0.05 vs. AG₅₀HT). ANOVA: analysis of variance; AG: aminoguanidine; HT: homocysteine thiolactone.

Aminoguanidine increased the incidence of HT-induced seizures in a dose-dependent manner. The seizure incidence was significantly increased in AG₁₀₀HT group in comparison with HT group (p < 0.01, Figure 1).

Time to first seizure episode onset (seizure latency) was significantly shorter in rats from AG₁₀₀HT group in comparison with those from HT group (p < 0.05, Figure 2(a)).

The number of seizure episodes per rat was significantly higher in groups AG₇₅HT (p < 0.05) and AG₁₀₀HT (p < 0.01) versus HT group. Furthermore, this number was significantly higher in AG₁₀₀HT compared to AG₅₀HT (p < 0.01, Figure 2(b)).

The same holds true for seizure severity in group AG₁₀₀HT in which seizure severity was significantly higher in comparison with HT and AG₅₀HT group (p < 0.01). Also, intensity of seizures was significantly higher in AG₅₀HT group compared to HT (p < 0.05, Figure 2(c)).

No lethality was recorded in HT and AG₅₀HT groups neither at 90 min nor 24 h after HT injection. Lethality was recorded in AG₇₅HT and AG₁₀₀HT in both time points. Significant differences were observed upon comparing these groups with HT and AG₅₀HT groups in the second time point, that is, 24 h after HT injection (Table 1).

Table 1.

The effects of aminoguanidine on lethality recorded 90 min and 24 h after HT administration.^a

Lethality^b (%)	Experimental groups
Lethality^b (%)	HT	AG₅₀HT	AG₇₅HT	AG₁₀₀HT
After 90 min	0	0	25.0	37.5
After 24 h	0	0	50.0^c,d	62.5^e,f

AG: aminoguanidine; HT: homocysteine thiolactone.

^aSignificance of the differences between the groups was estimated by Fisher’s exact probability test. For details see caption to Figure 1.

^bLethality—number of exited rats out of total rat number in group expressed in percentages.

^c p < 0.05.

^d p < 0.05.

^e p < 0.01 versus HT.

^f p < 0.01 versus AG₅₀HT.

EEG analysis

None of the epileptiform graphoelements was recorded on EEG of rats in C and AG₁₀₀ groups. Recordings of bioelectrical brain activity in rats from group C showed baseline activity (Figure 3(a)).

Figure 3.

Representative EEG tracings (left panels, amplitude scale 100 μV/div; timescale 1s/div) and corresponding power spectra density (right panels) recorded in control rats 30 min after saline treatment (a) and in AG₁₀₀HTgroup of rats 30 min after HT administration (b). Note baseline activity without epileptiform activity in (a) and SWD as typical ictal pattern characterized by paroxysmal, rhythmic 5–7 Hz SWDs with amplitude of at least twice the background EEG activity evident also as highly increased power spectral density (b). Quantitative analysis of SWD appearance included assessment of the number (c) and duration (d) of SWDs during 90 min after HT administration. The significance of the differences between the groups was estimated by Mann–Whitney U test (*p < 0.05). For details see caption to Figure 1. AG: aminoguanidine; HT: homocysteine thiolactone; SWDs: spike and wave discharges; EEG: electroencephalogram; div: division.

EEG recordings in rats from AG₁₀₀HT group showed ictal activity in the form of SWDs (Figure 3(b), left panel) accompanied with markedly elevated power spectral density (Figure 3(b), right panel). The median number of SWD per rat was significantly higher in AG₁₀₀HT compared to HT group (p < 0.05, Figure 3(c)). The same holds true for duration of SWD (Figure 3(d)).

Discussion

The results of this study showed that pretreatment with aminoguanidine, a selective iNOS inhibitor, increased convulsive properties, that is, seizure incidence, number of seizure episodes per rat, and severity of HT-induced seizures as well as number and duration of SWDs on EEG. Also, aminoguanidine decreased the latency time to the first seizure episode induced by HT in the same dose-dependent manner.

Mechanisms by which HT induces seizures and other neurotoxic effects are complex and not yet fully understood. Until today, there is evidence that many excitatory effects are the results of excessive glutamate (both ionotropic and metabotropic) receptors activation,²⁰ oxidative stress, inhibition of Na⁺/K⁺-ATPase activity,²¹ as well as DNA damage and induction of apoptosis.^4,6

We have recently showed the functional involvement of NO in the HT-induced convulsive activity, by demonstrating that l-arginine, NOS substrate, significantly decreased seizure incidence and prolonged latency time to the first seizure induced by a convulsive dose of HT.¹⁸ On the other hand, pretreatment with l-nitroarginine methyl ester (l-NAME) showed inverse results on seizures elicited by subconvulsive dose of HT. In the same study, l-arginine decreased and l-NAME increased the median number of SWD per rat, without significant effects on SWD duration. The involvement of nNOS in HT-induced seizures was determined using a pharmacological inhibition of this enzyme by 7-nitroindazole.²² Congruent results with those obtained using nonselective inhibition were obtained.

Pharmacological studies have used different nonselective (such as l-NAME) or selective inhibitors of NOS (mostly 7-nitroindazole or aminoguanidine for nNOS or iNOS inhibition, respectively) or its substrate (l-arginine) in order to understand the role of NO in epileptogenesis, that is, whether NO has pro- or anticonvulsive properties. Reported results are conflicting.

Anticonvulsive role of NO has also been shown in picrotoxin-induced convulsions²³ and penicillin-induced epileptiform activity in rats.²⁴ l-NAME caused an increase in both the seizure severity and duration in mice model of generalized epilepsy induced by N-methyl-d-aspartate (NMDA).²⁵ Tutka et al.²⁶ reported involvement of NO in convulsions induced by nicotine in mice.

In the case of pentylenetetrazole (PTZ), gamma- aminobutyric acid (GABA_A)-mediated convulsive agent, NOS inhibitors may inhibit convulsions.²⁷ However, recent study revealed that nNOS knockout mice (nNOS^−/ ⁻) exhibited severe convulsions following administration of subconvulsive dose of PTZ.²⁸ On the other hand, there is evidence showing no effect on PTZ-induced convulsions.²⁹ In line with these findings, treatment of pilocarpine-induced seizures with NOS inhibitors has been shown to potentiate,³⁰ to inhibit,³¹ or to be without effects³² on the epileptic activity.

On the other hand, it has been demonstrated that NO has proconvulsive activity in several seizure models (reviewed in the literature¹¹), like seizures induced by lindane, neurotoxin acting via GABA receptor.³³

Besides opposite effects of NO in different seizure models, it has been shown that the contribution of NO signaling in mechanisms of epileptogenesis depends on the source of NO, that is, whether it is produced by the activity of nNOS or iNOS.^12,34 Also, opposite responses of NOS to generalized seizures along the anterior–posterior axis of the brain have been recently proved.³⁵ Increase in iNOS expression has been found in brains of surgical patients with prolonged history of epilepsy,¹⁶ as well as in some spontaneously epileptic animals.¹⁷

Having in mind potential therapeutic applications, numerous attempts were also made to elucidate the effects of NOS inhibitors on the activity of different antiepileptic drugs. Recently, Adabi Mohazab et al.³⁶ showed that coadministration of aminoguanidine (100 mg/kg) significantly decreased the beneficial effects of pioglitazone on PTZ-induced seizures in mice. Bahremand et al.³⁷ using PTZ-induced seizures, showed that constitutive forms of NOS, but not iNOS, were involved in the anticonvulsant effect of lithium in this seizure model.

The majority of the pharmacological studies on functional involvement of NO in the development of epileptic events analyzed its motor phenomena, while not so many were focused on EEG activity. In this study, we applied both approaches. Assessment of convulsive behavior as well as quantitative analysis of ictal activity on EEG showed congruent results in the present study. Namely, aminoguanidine pretreatment significantly increased the number and duration of SWD induced by HT in this study, indicating increased hyperexcitability provoked by iNOS inhibition.

Although it has been hypothesized that overproduction of NO via stimulation of iNOS could contribute to inflammatory response, one possible mechanism in epileptogenesis, our data showed that selective inhibition of iNOS increased susceptibility for HT seizures.

Anticonvulsive role of iNOS-derived NO, demonstrated in this study, could be attributed to several mechanisms.

NO interacts with the redox site of the NMDA receptor complex and decreases its response to agonists, preferably by S-nitrosylation. This could be particularly important in situations of hyperexcitability and other pathological conditions.^38,39 Glutamate levels are significantly higher in the cortex, hippocampus, and brain stem of iNOS^−/− mice compared to the iNOS^+/+ mice.⁴⁰

It was found that high concentration of NO increases the GABA release.⁴¹ Thus, it is possible that NO level reduction could result in a depression of GABA-ergic system with hyperexcitability as a consequence. Moreover, evidence exists that NO could inhibit GABA transaminase.²³

It has been shown that NO is capable of achieving neuroprotective and antioxidative effects by building S-nitroso-l-glutathione,⁴² S-nitrosylation-mediated inhibition of caspase activity,⁴³ and promotion of the expression of cytoprotective genes.⁴⁴

With the emergence of reports on the neuroprotective effects of NO, the prior dogma about NO being solely detrimental has been modified. It is believed that the effects of NO depend on the environment and the context in which NO is produced.¹⁵

Based on the results of this study, it could be concluded that aminoguanidine, selective iNOS inhibitor, potentiates epilepsy induced by HT in subconvulsive dose, showing that iNOS-derived NO has anticonvulsive properties in the development of these seizures. Findings of this study may be of particular value for shedding more light on the pathophysiology of the neurological morbidities in patients developing hyperhomocysteinemia, as well as for the development of new treatment strategies for seizures in these patients.

Footnotes

Funding

This work was supported by the Ministry of Education, Science, and Technological Development of Serbia (grant number 175032).

Conflict of Interest

The authors declared no conflicts of interest.

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