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The Effect of Nitrendipine and Levetiracetam in Pentylenetetrazole Kindled Rats Meryem Dilek Karakurt1, Süleyman Emre Kocacan2, Cafer Marangoz3

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The Effect of Nitrendipine and Levetiracetam in Pentylenetetrazole Kindled Rats Meryem Dilek Karakurt1, Süleyman Emre Kocacan2, Cafer Marangoz3 Meryem Dilek Karakurt et al., IJNR, 2019; 3:9 Research Article IJNR (2019) 3:9 International Journal of Neuroscience Research (ISSN:2572-8385) The Effect of Nitrendipine and Levetiracetam in Pentylenetetrazole Kindled Rats Meryem Dilek Karakurt1, Süleyman Emre Kocacan2, Cafer Marangoz3 1Ankara Yıldırım Beyazıt University Medical Faculty, Ankara, TURKEY 2Ondokuz Mayıs University Medical Faculty, Samsun, TURKEY 3Istanbul Medipol University Medical Faculty, Istanbul, TURKEY ABSTRACT We aimed to investigate the efficacy of L-type voltage gated Abbrevations: calcium channel blocker nitrendipine and levetiracetam in PTZ: Pentylenetetrazol; DMSO: pentylenetetrazole (PTZ) kindled male rats. In order to establish Dimethyl sulfoxide; sc: Second; kindling model, 35 mg/kg PTZ injected intraperitoneally (i.p.) EEG: Electroencephalography; to male wistar albino rats three days a week. Then, screw mV: Milivolt; electrodes were placed in the skulls of the kindled rats. During the experiments, EEG activities and seizure behaviors of kindled *Correspondence to Author: rats were recorded. The kindled rats were divided into control Meryem Dilek Karakurt (n=6), PTZ (n=6), nitrendipine (2.5 mg/kg (n=6), 5 mg/kg (n=6), Ankara Yıldırım Beyazıt University 10 mg/kg (n=6)) and levetiracetam (10 mg/kg (n=6), 20 mg/ Medical Faculty, Physiology De- kg (n=6), 40 mg/kg (n=6)) groups. Nitrendipine (5 mg/kg) and partment, 06010 Ankara, TURKEY levetiracetam (20 mg/kg) were suppressed the spike frequency and the seizure score effectively (p<0.05). The effective doses How to cite this article: of nitrendipine (5 mg/kg) and levetiracetam (20 mg/kg) were Meryem Dilek Karakurt, Süleyman administered consecutively to the kindled animals (n=6). The Emre Kocacan, Cafer Marangoz. co-administration of nitrendipine (5 mg/kg) and levetiracetam The Effect of Nitrendipine and Le- (20 mg/kg) did not effectively decrease the spike frequency and vetiracetam in Pentylenetetrazole seizure score (p>0.05). The co-administration of nitrendipine Kindled Rats. International Journal and levetiracetam was not more effective than administration of of Neuroscience Research, 2019; nitrendipine or levetiracetam separately (p>0.05). 3:9. Keywords: levetiracetam, nitrendipine, kindling model, voltage- gated calcium channels, rat eSciPub LLC, Houston, TX USA. Website: https://escipub.com/ IJNR: https://escipub.com/international-journal-of-neuroscience-research/ 1 Meryem Dilek Karakurt et al., IJNR, 2019; 3:9 1. Introduction According to The Report of the American Epilepsy Society and the Guideline Kindling is the progressive development of Development as a result of Class I studies, seizures in response to administration of a levetiracetam is effective as add-on therapy for subconvulsant stimulus in a repeated fashion [1, treatment of resistant generalize epilepsy with 2] and it produces circuitry seen in complex generalize tonic clonic seizures and also add- partial seizures with secondary generalization on therapy for treatment of juvenile myoclonic [3]. The reorganizations which are the epilepsy and juvenile absence seizures [17]. It responses to excess neuronal activity, includes has been known that levetiracetam binds the transcription factors, immediate early genes, synaptic vesicle protein 2A [18], the activity of protein synthesis, neurogenesis, and which is related to the exocytosis of transmitter synaptogenesis, in addition to activation of vesicles [19, 20]. Several studies investigated the glutamate receptors, second messengers, anticonvulsant effect of levetiracetam via neurotrophic factors, and axon guidance voltage gated calcium channels [21, 22]. To molecules [4]. The electrical kindling and the determine the efficacy of nitrendipine and chemical kindling are two main kinds of the levetiracetam that both have effect on seizure kindling model. Subconvulsant chemical and suppression via voltage gated calcium channels subconvulsant electrical stimulation appear to the experiments exhibited in awake kindled rats activate similar anatomic pathways, however using simultaneous observations of chemical kindling affects the whole brain for a electrophysiological activity and seizure relatively long period. As a chemoconvulsant behavior. pentylenetetrazole (PTZ) is one of the most preferred substances used in the development 2. Materials and Methods of chemical kindling [3]. PTZ is a noncompetitive 2.1. Animals antagonist, which blocks gamma aminobutyric Male Wistar albino rats (12–16 weeks, weighing acid (GABA)-mediated Cl- influx through Cl- 200 ± 25 g) were used. The rats were housed [5] channels . individually in a 12 h light, 12 h dark cycle at a Large elevations in neuronal calcium, especially temperature of 22±2° C and 50–55% moisture. via L-type voltage-gated calcium channels, are Water and food were provided ad libitum. This mainly responsible for seizure generation and study was approved by the local Ethical propagation of epileptic discharges [6, 7]. Committee for Animal Experiments at the Nitrendipine is a dihidropyridine analog which University of Ondokuz Mayis in Samsun. All acts as L-type voltage-gated calcium channel experiments were conducted according to the blocker [8]. Nitrendipine binds the α1 subunit of guidelines of the European Community Council L-type voltage-gated calcium channels [9] for animal care. responsible for the opening and closing of the 2.2. Kindling channel [10]. Nitrendipine can cross the blood To induce kindling, the rats were injected i.p. brain barrier because of its highly lipophilic with a subconvulsive dose of PTZ (35 mg/kg) structure [11]. Although, nitrendipine is an three times a week (Monday, Wednesday, and antihypertensive drug used for primary Friday). After the injections the animals were hypertension [8, 12], the potential application of placed individually in transparent boxes (35 dihidropyridine analogs in a range of areas has (L)×35 (W)×35 (H) cm). Their seizure behaviors been studied to determine whether it offers were observed for 30 minutes (min) according advantages compared to current therapies [13]. to Fischer and Kittner’s seizure scale of 1–5 Also, the anticonvulsant activity of nitrendipine and stage 3–5 seizure behaviors were has been investigated in a variety of seizure accepted as generalized seizure. Stages were models [6, 14-16]. IJNR: https://escipub.com/international-journal-of-neuroscience-research/ 2 Meryem Dilek Karakurt et al., IJNR, 2019; 3:9 characterized as follows. Stage 0: no seizure. the EEG recordings and the spike frequency for Stage 0.5: weak nodding. Stage 1: twitching of each animal were automatically calculated face, eyelids, and ears. Stage 1.5: mild clonic using the Chart v.5.1.1 program. activity of forelimb. Stage 2: myoclonic body 2.5. Drugs and Routes jerks, clonic movement of forelimb without Drugs were administered as follows: rearing. Stage 2.5: swift clonic seizures of forelimb following partial rearing. Stage 3: PTZ: Dissolved in saline, injected i.p. robust bilateral forelimb clonus with complete Nitrendipine: Dissolved in 3% DMSO, rearing (≥ 10 sec). Stage 3.5: rearing and falling administered i.p. with intense bilateral forelimb clonus. Stage 4: Levetiracetam: Dissolved in saline, generalized clonic seizures with rearing-falling administered i.p. down episodes or jumping. Stage 4.5: PTZ, nitrendipine, and levetiracetam were generalized clonic-tonic seizures with failure of purchased from Sigma-Aldrich Co. righting reflex. Stage 5: generalized clonic-tonic 2.6. Experimental Groups seizures and status epilepticus (≥2 min). The rats were kindled after having five generalized The kindled rats (n=60) were divided into 10 seizures [23]. To induce kindling, injections were groups. The groups were as follows. performed over a period of four weeks [24]. Control group: EEGs and seizure stages Nonkindled and dead rats were excluded from recorded without administration of any drug for the study. 30 min (n=6). 2.3. Surgical Operation PTZ group: PTZ 35 mg/kg (n=6). The kindled rats were anesthetized with 90 Nitrendipine groups: Nitrendipine 2.5 mg/kg mg/kg ketamine hydrochloride and 10 mg/kg (n=6), 5 mg/kg (n=6), and 10 mg/kg (n=6) 30 xylasine (i.p.). Then rats positioned on a min before PTZ injection. stereotaxic apparatus (Harvard). After a midline Levetiracetam groups: Levetiracetam 10 mg/kg scalp incision, stainless steel screw electrodes (n=6), 20 mg/kg (n=6), and 40 mg/kg (n=6) 30 were fixed into the skull by the following min before PTZ injection. coordinates: first electrode: 3 mm lateral to Nitrendipine-Levetiracetam group: Nitrendipine sagittal suture, 4 mm anterior to Bregma at a dose of 5 mg/kg and levetiracetam at a (primary motor cortex, M1); and second dose of 20 mg/kg were injected consecutively electrode: 3 mm lateral to sagittal suture, 4 mm (i.p.) into rats 30 min before PTZ injection posterior to Bregma (medial parietal association (n=6). cortex, MPtA) [25]. An electrode was identified as a reference and positioned in the skull at 4 DMSO group: 3% DMSO (a volume of 0.1 ml) mm posterior to the Bregma. Electrodes fixed in was injected (i.p.) into rats 30 min before PTZ the skull were attached to a connecting socket. injection (n=6). Except the socket, the electrodes on the 2.7. Statistical Analysis surface of the skull were implanted by using The data obtained were assessed using the cold-cured dental acrylic. SPSS package program. Shapiro–Wilk and 2.4. Electrophysiological Recordings Kolmogorov–Simirnov tests were used for After the surgery recovery period of a week, determining the normal distribution of the awake kindled rats were connected to a variables. A one-way analysis of variance computerized EEG recording system (ANOVA) and the post-hoc Tukey test were (PowerLab/4SP, AD Instruments). The seizure used for comparing the spike frequency data behaviors of the animals were observed at the among the groups. The seizure stage data same time for 30 min. The off-line analysis of were analyzed with the Kruskal–Wallis test, and IJNR: https://escipub.com/international-journal-of-neuroscience-research/ 3 Meryem Dilek Karakurt et al., IJNR, 2019; 3:9 the Mann–Whitney U Test was used for binary After the PTZ (35 mg/kg, i.p.) injection, EEG comparison.
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