Research Article

The Protective Effects of Vitamin B₁₂ on Pentylenetetrazole-Induced Seizures in Rats

Ahmet Sevki Taskıran¹*, Erkan Gumus², Handan Gunes¹, Arzuhan Cetindag¹, Ercan Ozdemir¹ and Gokhan Arslan¹

¹Department of Physiology, Cumhuriyet University School of Medicine, Turkey

²Department of Histology and Embryology, Cumhuriyet University School of Medicine, Turkey

Submission: December 07, 2017; Published: January 19, 2018

*Corresponding author: Ahmet Sevki Taskıran, Department of Physiology, Cumhuriyet University School of Medicine, Sivas, Turkey, Tel: +90-346-219-1010-2915; Fax: +90-346-219-1602; Email: a.sevkitaskiran@gmail.com

How to cite this article: Ahmet Sevki Taskıran*, Erkan Gumus, Handan Gunes, Arzuhan Cetindag, Ercan Ozdemir and Gokhan Arslan. The Protective Effects of Vitamin B₁₂ on Pentylenetetrazole-Induced Seizures in Rats. Anatomy Physiol Biochem Int J: 2018; 4(1): 555627. DOI: 10.19080/APBIJ.2018.04.555627.

Abstract

Objective: Epilepsy is defined as a short-lived paroxysmal disorder of the brain functions observed in seizures by sudden, abnormal and hypersynchronous discharges of a group of neurons in the central nervous system. Vitamin B₁₂ derivatives are complex organometallic cofactors used by a limited number of enzymes. B₁₂ vitamins are involved in many cellular functions, both glial and neuronal, in the central and peripheral nervous system. The aim of this study was investigate to effects of vitamin B₁₂ on pentylenetetrazole-induced seizures in rats.

Methods: In our study, 18 240-280gr male Wistar albino rats were used. Animals were divided into three groups: control (n=6), 50μg/kg vitamin B₁₂ (n=6) and 100μg/kg vitamin B₁₂ (n=6). Serum physiologic to first group and other two groups were administered for seven days at the indicated doses of vitamin B₁₂ intraperitoneally. On the seventh day, pentylenetetrazole (PTZ) was intraperitoneally injected at 70mg/kg 45 minutes after drug administration. The animals were observed for 30 min. Stages were determined according to the Racine seizure scale and the first myoclonic jerk time (FMJ) was recorded in seconds. After the procedure, brain tissues were removed of animals. After routine histological follow-up, serial sections from brain tissues were stained with toluidine blue. The hippocampal CA1, CA2 and dentate gyrus regions were evaluated histopathologically. The data analyses were performed with SPSS Version 21.0 for Windows and were evaluated using a one-way analysis of variance (ANOVA).

Results: The results of epileptic behavior were evaluated according to Racine convulsion scale, the difference between the control and 50μg vitamin B₁₂ group was statistically significant (p<0.01). The first myoclonic jerk time was considered, the difference between the control and 50μg vitamin B₁₂ was statistically significant (p<0.001). When the groups were evaluated histopathologically, it was statistically significant that 50μg B₁₂ treatment reduced neuronal damage in CA1, CA2 and dentate gyrus regions (p<0.05).

Conclusion: This study suggests that vitamin B₁₂ therapy may reduce epileptic seizures and post-seizure neuronal damage.

Abbreviations: PTZ: Pentylene Tetrazole; FMJ: First Myoclonic Jerk; AEDs: Antiepileptic Drugs; RCS: Racine’s Convulsion Scale; CA: Cornu Ammonis; DG: Dentate Gyrus; SEM: Standard Error of Mean

Keywords: Epilepsy; Pentylenetetrazole; Vitamin B₁₂

Introduction

Epilepsy is the one of most common and critical neurological disorder that affects millions of people worldwide [1]. This neurological disorder is defined the repeated occurrence of bursts of electrical activity (seizures) in specific brain areas such as limbic system and cerebral cortex [2]. Various antiepileptic drugs (AEDs) are widely used both long-term combined therapy and mono therapy in epilepsy. Drug resistant (i.e., pharmacoresistant or medically intractable) epilepsy is defined as failure to achieve seizure control despite adequate trials of antiepileptic drug (AED) therapy [3] and approximately one-third of epileptic patients do not respond efficiently to present AEDs [4]. Many available AEDs may also cause toxicity [5]. Therefore, more influential and safer new therapeutics are necessary.

Vitamin B₁₂ is needed for the development and initial myelination of the central nervous system as well as for the maintenance of its normal function [6]. Promoting neurite growth, neuroregeneration and antinociception properties of vitamin B₁₂ were studied in several animal models related to neuronal diseases [7,8]. Neurological problems arise from vitamin B₁₂ deficiency have wide spectrum, from asymptomatic to life-threat pancytopenia or myelopathy. It was also implicated that vitamin B₁₂ is a risk factor in the etiology of ischemic vascular events and in the course of many degenerative diseases, particularly Alzheimer’s disease [9,10]. In addition, vitamin B₁₂ deficiency has been shown to cause epileptic seizures [11,12]. Although there are enhancement evidences indicating neuroprotective effect of vitamin B₁₂, the effect of vitamin B₁₂ on the antiepileptic effect has not been clearly demonstrated. This study was designed to investigate the effect of vitamin B₁₂ on pentylenetetrazole-induced seizures and to demonstrate the neuroprotective effect of vitamin B₁₂ on neuronal damage after pentylentetrazol administration.

Materials and Methods

Experimental animals

The present study was performed in Cumhuriyet University Animal Laboratory after approval of Local Ethics Committee. Healthy adult male, weighing 230-250 g, Wistar albino (n=18) were used. All animals were fed by a standard laboratory diet and they could drink water whenever they requested. Rats were capable of normal activity in the cages, 22 ± 2°C, humidity (50- 70%) and 12 hours of night/day. All animals were kept under observation for a few days before the study to decide if they are healthy or not.

Drug administration

Vitamin B₁₂ and pentylentetrazol were dissolved in physiological saline. The drugs were purchased from Sigma- Aldrich Co., St Louis, MO, USA. Solutions were freshly prepared on the days of the experiments.

Experimental procedure

Eighteen rats were divided randomly into three groups for behavioral and histological assessments. Group1 was given saline intraperitoneally (i.p.), group2 50μg/kg vitamin B₁₂ i.p. and group3 100μg/kg vitamin B₁₂ i.p. for 7 days. The seventh day, pentylentetrazol (PTZ) (70 mg/kg, i.p.) was injected 45 min after last vitamin B₁₂ injection to induce seizures. Racine’s Convulsion Scale (RCS) were used to evaluate the seizures stages 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. Rats were observed for both to evaluate Racine’s Convulsion Scale (RCS) and to record first myoclonic jerk(FMJ) onset times which coincide inception stage3 [13]. The observation period for PTZinduced seizures was limited to 30 min in duration [14]. Two hours after, the animals were terminated using the decapitation method and brain tissues were removed.

Histopathological evaluation

Formalin-fixed brain sections (5μm) were stained with toluidine blue stain to quantify the number of dark neurons. All sections were examined and photographed with Olympus C-5050 digital camera at Olympus BX51 microscope. In hippocampal CA1, CA2 (Cornu Ammonis) and DG (Dentate gyrus) regions, dark neurons and survival neurons were counted in six sections per studied animal (n=3 for each group) by an image analysis system (Image- Pro Express 1.4.5, Media Cybernetics, Inc. USA). The numbers of dark neurons were given as percentage (toluidine blue stained neurons*100/survival neuron). The observers blinded to the study groups accomplished all histological assessments.

Statistical analysis

The results were expressed as a mean ± standard error of mean (SEM). The data analyses were performed with SPSS Version 21.0 for Windows. The RCS score, FMJ time and dark neurons were evaluated using a one-way analysis of variance (ANOVA). A posthoc Tukey test was utilized to identify the differences between the experimental groups, and a value of p < 0.05 was accepted as statistically significant.

Results

Evaluation of groups in terms of RCS and FMJ Onset Times

When the Racine scores were calculated between the groups, there were statistically significant differences between the saline (5.5 ± 0.2) and 50μg/kg vitamin B₁₂ (3.6± 0.4)(p <0.01). However there were no statistically significant differences between the saline (5.5 ± 0.2) and the 100μg/kg vitamin B₁₂ (4.5 ± 0.4) group than in the saline (p >0.05). In addition, there were no differences between the 50μg/kg vitamin B₁₂ and 100μg/kg vitamin B₁₂ (p > 0.05) (Figure 1). There were statistically significant differences (p < 0.001) between saline (83.3 ± 9.9 s) and 50μg/kg vitamin B₁₂ (176.6 ± 16.7 s) groups in terms of FMJ onset times. There were no statistically significant differences between 100μg/kg vitamin B₁₂ (109.3± 11.2) and saline in terms of FMJ onset times (p>0.05). In addition, there were differences between 50μg/ kg vitamin B₁₂ and 100μg/kg vitamin B₁₂ statistically (p<0.01) (Table 1).

Evaluation of groups in terms of dark neurons

Dark neurons were identified by the neuronal shrinkage, cytoplasmic esoinophilia, nuclear pyknosis, and surrounding spongiosis in total hippocampal formation (Figure 2A,2E,2I) and CA1 (Figure 2B,2F,2J), CA2 (Figures 2C,2G,2K) and DG (Figures 2D,2H,2L) hippocampal regions’ formation. Administration of Vitamin B₁₂ at the dose of 50μg/kg significantly prevented production of dark neurons due to PTZ induced seizures in CA1, CA2 and DG regions of hippocampus (p< 0.05) (Figures 3A-3C). However there was no significant difference between at the dose of of 100μg/kg vitamin B₁₂ and PTZ group in CA1, CA2 and DG regions of hippocampus in point of dark neurons (p>0,05) (Figures 3A-3C). In addition, there was no significant difference between at the dose of 50μg/kg and at the dose of 100 μg/kg vitamin B₁₂ in CA1, CA2 and DG regions of hippocampus in point of dark neurons (p>0,05).

Discussion

In the present study, on the one hand subchronic administration of vitamin B₁₂ significantly decreased RC and also increased FMJ. Vitamins have been considered important patterns in controlling certain types of seizures or even preventing adverse effects of AEDs [15,16]. There are various studies indicating an association between vitamin B₁₂ deficiency and EEG abnormalities in epilepsy [17,18]. In addition, there are increasing evidences indicate neuroprotective effects of vitamin B₁₂ in peripheral and central nervous systems. In the previous studies, researchers evaluated the neuroprotective actions of vitamin B₁₂, in rats with sciatic and corneal nerves crush injury models [19,20]. In addition, a study indicated that vitamin B₁₂ has anti-apoptotic effect on peripheral neuron injury by increasing Bax protein and reducing Bcl-2 protein [21]. Furthermore, studies have shown that vitamin B₁₂ is able to protect cortical neurons and retinal cell cultures against glutamate cytotoxicity [22]. Also vitamin B₁₂ show antiepileptic activity in penicillininduced model via GABAA receptor system [23]. On the other hand, neuroprotection is very important as a promising therapy for preventing and treating epilepsy [24]. These findings show possibility potential of vitamin B₁₂ in the treatment epilepsy. However, the potential use of vitamin B₁₂ in the treatment of epilepsy is not enough yet.

On the other hand, these findings show possibility potential of vitamin B₁₂ in the treatment epilepsy. However, the potential use of vitamin B₁₂ in the treatment of epilepsy is not enough yet. In present study showed that after PTZ-induced seizures damaged to hippocampals neurons of the rats were prevented by vitamin B₁₂. Dark neurons, previously were considered as histological artifacts in neuro surgical biopsies [25] but later, they were seen after brain trauma [26].

Dark neurons have basophilic appearance and morphological changes and might be seen after hypoglycemia, ischemia, stress and epilepsy [27,28]. Epilepsy has also been introduced as an important cause of dark neuron production [29]. The results of present study showed that PTZ-induced seizures were resulted in dark neuron production in the hippocampal regions which were confirmed by various studies [30,31]. Several studies have also confirmed hippocampal damages created by seizures [32,33]. The results of present study were consistent with previous studies in which it was shown that PTZ-induced seizures were followed by production of dark neurons in the brain tissues.

Conclusion

The results of the present study showed that vitamin B₁₂ decreased epileptic seizures as well as preventing neural damage after PTZ- induced seizure in rats. These results support the beneficial effect vitamin B₁₂ on the nervous system. Further studies are required for determining the protective effect and mechanism of vitamin B₁₂.

Financial Disclosure

No funding agency had any part in this study.

Conflict of Interest

The authors declare no competing interests.

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