Investigation of the Effect of Diazepam on Nitric Oxide Synthase and Brain-Derived Neurotrophic Factor in Pentylenetetrazole-Induced Acute Seizure Model

Abstract

The aim of this study was to investigate the effect of diazepam on nitric oxide synthase (NOS) and brain-derived neurotrophic factor (BDNF) in the anti-convulsant activity of diazepam on pentylenetetrazole (PTZ) induced seizures in rats. 24 male Wistar Albino rats were used in the study. Rats were divided into 4 groups as control group, saline (1 mL kg-1) +PTZ, diazepam (0.5 mg kg-1) +PTZ and diazepam (2 mg kg-1) +PTZ group (n=6). For biochemical studies, 24 hours after PTZ application, brain tissues of all rats were removed and cortex and hippocampus regions were isolated NOS and BDNF levels in the cortex and hippocampus were measured using ELISA kits. Diazepam significantly decreased the seizure stage and prolonged the first myoclonic jerk (IMJ) time compared to the saline group (p<0.001). Saline+PTZ significantly decreased NOS levels in the hippocampus when compared to the control group (p<0.05). However, the diazepam+PTZ groups significantly increased the hippocampal NOS level compared to the saline+PTZ group (p<0.001). In addition, the saline + PTZ group increased the BDNF level in the cortex and hippocampus compared to the control (p<0.05), while the BDNF level was lower in the diazepam+PTZ group compared to the saline+PTZ group (p<0.001). Diazepam increased NOS levels in the hippocampus and decreased BDNF levels in the cortex and hippocampus. In conclusion, the regulatory effect of diazepam on NOS and BDNF may be related to its anti-convulsant mechanisms.

Keywords

• Seizure
• Pentylentetrazole Diazepam
• Nitric Oxide Syntase
• Brain Derived Neurotrophic Factor

Pentilentetrazol ile Oluşturulan Akut Nöbet Modelinde Diazepamın Nitrik Oksit Sentaz ve Beyin Kaynaklı Nörotrofik Faktör Üzerine Etkisinin Araştırılması

Öz

Bu çalışmanın amacı, sıçanlarda pentilentetrazol (PTZ) ile oluşturulan nöbetlerde diazepamın antikonvulzif etkinliğinde nitrik oksit sentaz (NOS) ve beyin kaynaklı nörotrofik faktör (BDNF) üzerine etkisini araştırmaktır. Çalışmada 24 adet erkek Wistar Albino sıçan kullanılmıştır. Sıçanlar kontrol grubu, salin (1 mL kg-1) + PTZ, diazepam (0,5 mg kg-1) + PTZ ve diazepam (2 mg kg-1)+PTZ grubu olarak 4 gruba ayrılmıştır (n=6). İntraperitoneal ilaç uygulamalarından 24 saat sonra biyokimyasal incelemeler için tüm sıçanların beyin dokuları çıkarılıp, korteks ve hipokampüs bölgeleri ayrılmıştır. Korteks ve hipokampüsteki NOS ve BDNF düzeyleri enzim bağlı immünosorbent analizi (ELISA) kitleri kullanılarak ölçülmüştür. Diazepam, salin+PTZ uygulanan grubuna göre istatistiksel açıdan anlamlı bir şekilde nöbet evresini azalttı ve ilk miyoklonik jerk (İMJ) süresini uzattı (p<0.001). Salin+PTZ, kontrol grubuna kıyasla hipokampüste NOS seviyelerini anlamlı olarak azalttı (p<0.05). Bununla birlikte diazepam+PTZ grupları, salin+PTZ grubuyla karşılaştırıldığında hipokampal NOS seviyesini anlamlı olarak artırdı (p<0.001). Ayrıca,salin+PTZ grubu kontrolle karşılaştırıldığında korteks ve hipokampüste BDNF seviyesini artırırken (p<0.05), diazepam+PTZ uygulanan gruplarda BDNF seviyesi salin+PTZ grubuna kıyasla düşüktü (p<0.001). Diazepam hipokampüste NOS seviyesini artırmış, korteks ve hipokampüste ise BDNF seviyelerini düşürmüştür. Sonuç olarak, diazepamın NOS ve BDNF üzerine regülatif etkisi antikonvulsif etkisiyle ilişkili olabilir.

Anahtar Kelimeler

• Nöbet
• Pentilentetrazol (PTZ)
• Diazepam
• Nitrik Oksit Sentaz (NOS)
• Beyin Kaynaklı Nörotrofik Faktör (BDNF)

Kaynakça

1. [1] Thomas R. Browne, Gregory L. Holmes. Epilepsy: Definitions and Background. Hanbook of Epilepsy. Third edition. USA; 2004 .p. 6-7.
2. [2] Baykan B, Bebek N, Candan Gürses, Gökyiğit A. Epilepsi. Ed: Öge AE, Baykan B. Nöroloji. 2.Baskı. İstanbul: Nobel Tıp Kitabevleri; 2004. s. 279-309.
3. [3] Gunes H, Ozdemir E, Arslan. Coenzyme Q10 increases absence seizures in WAG/Rij rats: The role of the nitric oxide pathway. Epilepsy Res. 2019 Aug;154:69-73.
4. [4] Zienowicz M, Wisłowska A, Lehner M. The effect of fluoxetine in a model of chemically induced seizures behavioral and immunocytochemical study. NeurosciLett. 2005; 373(3):226–231.
5. [5] E. Erkec, O. Arihan. Pentylenetetrazole kindling epilepsy model, Epilepsi. 2015; 21 (1) : 6–12.
6. [6] Meierkord H, Boon P, Engelsen B, Gocke K, Shorvon S, Tinuper Holtkamp M. EFNS guideline on the management of status epilepticus. Eur. J. Neurol. 2006;13: 445–450.
7. [7] Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, Mohler H, Benzodiazepine actions mediated by specific γ- aminobutyric acid (A) receptor subtypes. Nature.1999; 401:796–800.
8. [8] Hanson SM, Czajkowski C. Structural mechanisms underlying benzodiazepine modulation of the GABA(A) receptor. J. Neurosci. 2008; 28:3490–3499.

9. [9] Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 1991; 43: 109–142.
10. [10] Nidhi G, Balakrishnan S, Pandhi P. Role of nitric oxide in electroshock and pentylenetetrazole seizure threshold in rats. Methods Find. Exp. Clin. Pharmacol. 1999;21: 609–612.
11. [11] Rajasekaran K, Jayakumar R, Venkatachalam K. Increased neuronal nitric oxide synthase (nNOS) activity triggers picrotoxin-induced seizures in rats and evidence for participation of nNOS mechanism in the action of antiepileptic drugs. Brain Res. 2003; 979: 85–97.
12. [12] Lizasoain I, Knowles RG, Moncada S. Inhibition by lamotrigine of the generation of nitric oxide in rat forebrain slices. J. Neurochem.1995; 64: 636–642.
13. [13] Nidhi G, Balakrishnan S, Pandhi P. Role of nitric oxide in electroshock and pentylenetetrazole seizure threshold in rats. Methods Find. Exp. Clin. Pharmacol.1999; 21: 609–612.
14. [14] Talarek S, Fidecka S. Involvement of nitricoxidergic system in the hypnotic effects of benzodiazepines in mice. Pol. J. Pharmacol. 2004;56: 719–726.
15. [15] Jimenez-Velazquez G, Lopez-Munoz FJ, Fernandez-Guasti A. Participation of the GABA/benzodiazepine receptor and the NO-cyclicGMP pathway in the “antinociceptive-like effects” of diazepam. Pharmacol. Biochem. Behav. 2008; 91:128–133.
16. [16] Talarek S, Fidecka S. Role of nitric oxide in anticonvulsant effects of benzodiazepines in mice. Pol. J. Pharmacol. 2003; 55: 181–191.
17. [17] Lee FS, Chao MV. Neurotrophic factors. Neural Sciences. 2008; 1: 96-102.
18. [18] Dugich-Djordjevic MM, Peterson C, Isono F, Widmer HR, Denton TL, Bennett GL, Hefti F. Immunohistochemical visualization of brain-derived neurotrophic factor in the rat brain. European Journal of Neuroscience. 1995; 7: 1831-1839.
19. [19] Phillips HS, Hains JM, Laramee GR, Rosenthal A, Winslow JW. Widespread expression of BDNF but not NT3 by target areas of basal forebrain cholinergic neurons. Science. 1990; 250: 290- 294.
20. [20] Huntley GW, Benson DL, Jones EG, Isackson PJ. Developmental expression of brain derived neurotrophic factor mRNA by neurons of fetal and adult monkey prefrontal cortex. Brain Research Developmental Brain Research. 1992;70: 53-63.
21. [21] Nawa H, Carnahan J, Gail C. BDNF protein measured by a novel enzyme immunoassay in normal brain and after seizure: partial disagreement with mRNA levels. European Journal of Neuroscience. 1995; 7: 1527- 1535.
22. [22] Wetmore C, Cao Y, Pettersson RF, Olson L. Brain-derived neurotrophic factor: subcellular compartmentalization and interneuronal transfer as visualised with antipeptide antibodies. Proceedings of the National Academy of Sciences. 1991;88: 9843-9847.
23. [23] Albertson TE, Peterson SL, Stark LG. The anticonvulsant effects of diazepam and phenobarbital in prekindled and kindled seizures in rats. Neuropharmacology 1981; 20:597–603.
24. [24] Taskiran AS, Tastemur Y. The role of nitric oxide in anticonvulsant effects of lycopene supplementation on pentylenetetrazole-induced epileptic seizures in rats. Exp Brain Res. 2021;239(2):591-599.
25. [25] Ernst O, Zor T. Linearization of the Bradford protein assay. J Vis Exp. 2010; 12(38):1918.
26. [26] Sefil F, Kahraman I, Dokuyucu R, Gokce H, Ozturk A, Tutuk O, Aydin M, Ozkan U, Pinar N. Ameliorating effect of quercetin on acute pentylenetetrazole induced seizures in rats. Int J Clin Exp Med. 2014 Sep 15;7(9):2471-7.
27. [27] Gholipour T, Rasouli A, Jabbarzadeh A, Nezami BG, Riazi K, Sharifzadeh M, Dehpour AR. The interaction of sildenafil with the anticonvulsant effect of diazepam. Eur J Pharmacol. 2009 Sep 1;617(1-3):79-83.
28. [28] Mante PK, Adongo DW, Woode E. Anticonvulsant effects of antiaris toxicaria aqueous extract: investigation using animal models of temporal lobe epilepsy. BMC Res Notes.2017; 10: 167.
29. [29] Ferraro G, Sardo P. Nitric oxide and brain hyperexcitability. In Vivo. 2004; 18:357–36615.
30. [30] Watanabe M, Miyai A, Danjo S, Nakamura Y, Itoh K. The threshold of pentylenetetrazole-induced convulsive seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains. Exp Neurol. 2013;247:645-52.
31. [31] Gotti S, Sica M, Viglietti-Panzica C, Panzica G. Distribution of nitric oxide synthase immunoreactivity in the Mouse brain. Microsc Res Tech. 2005;68(1):13-35.
32. [32] Naziroğlu M, Kutluhan S, Yilmaz M. Selenium and topiramate modulates brain microsomal oxidative stress values, Ca2+-ATPase activity, and EEG records in pentylentetrazol-induced seizures in rats. J Membr Biol. 2008 Sep-Oct;225(1-3):39-49.
33. [33] Kapucu A. Effect of erythropoıetın on nıtrıc oxıde synthase types ın the hıppocampus and frontal cortex ın ptz ınduced seizure ın rats. Journal of Istanbul Faculty of Medicine. 2019;82(2):107-115.
34. [34] Mazhar F, Malhi SM, Simjee SU. Comparative studies on the effects of clinically used anticonvulsants on the oxidative stress biomarkers in pentylenetetrazole-induced kindling model of epileptogenesis in mice. J Basic Clin Physiol Pharmacol. 2017 Jan 1;28(1):31-42.
35. [35] Volke V, Soosaar A, Kõks S, Vasar E, Männistö PT. L-Arginine abolishes the anxiolytic-like effect of diazepam in the elevated plus-maze test in rats. Eur J Pharmacol. 1998 Jun 26;351(3):287-90.
36. [36] Standaert DG. NMDA receptors and nitric oxide synthase. Nature. 1999;4:13-14.
37. [37] Lucini C, D’Angelo L, Cacialli P, Palladino A, de Girolamo P. BDNF, brain, and regeneration: Insights from zebrafish. Int J Mol Sci. 2018; 19(10): 3155.
38. [38] Autry AE, Monteggia LM. Brain-derived neurotrophic factor and neuropsychiatric disorders. Pharmacol Rev. 2012;64(2):238-58.
39. [39] Hong Z, Li W, Qu B, Zou X, Chen J, Sander J, et al. Serum brain derived neurotrophic factor levels in epilepsy. Eur J Neurol 2014;21(1): 57-64.
40. [40] Altar CA, Whitehead RE, Chen R, Wörtwein G, Madsen TM. Effects of electroconvulsive seizures and antidepressant drugs on brain-derived neurotrophic factor protein in rat brain. Biol Psychiatry. 2003; 54(7): 703-9.
41. [41] Falcicchia C, Paolone G, Emerich DF, Lovisari F, Bell WJ, Fradet T, et al. Seizure-suppressant and neuroprotective effects of encapsulated BDNF-producing cells in a rat model of temporal lobe epilepsy. Mol Ther Methods Clin Dev. 2018; 9: 211-24.
42. [42] Porcher C, Medina I, Gaiarsa J-L. Mechanism of BDNF modulation in GABAergic synaptic transmission in healthy and disease brains. Front Cell Neurosci. 2018; 12: 273.
43. [43] Malhi SM, Jawed H, Hanif F, Ashraf N, Zubair F, Siddiqui BS, Begum S, Kabir N, Simjee SU. Modulation of c-Fos and BDNF protein expression in pentylenetetrazole-kindled mice following the treatment with novel antiepileptic compound HHL-6. Biomed Res Int. 2014;2014:876712.
44. [44] Şahin S, Gürgen SG, Yazar U, İnce İ, Kamaşak T, Acar Arslan E, Diler Durgut B, Dilber B, Cansu A. Vitamin D protects against hippocampal apoptosis related with seizures induced by kainic acid and pentylenetetrazol in rats. Epilepsy Res. 2019 Jan;149:107-116.
45. [45] Licata SC, Shinday NM, Huizenga MN, Darnell SB, Sangrey GR, Rudolph U, Rowlett JK, Sadri-Vakili G. Alterations in brain-derived neurotrophic factor in the mouse hippocampus following acute but not repeated benzodiazepine treatment. PloS One. 2013 Dec; 19:8(12):e84806.
46. [46] Regner GG, Torres ILS, de Oliveira C, Pflüger P, da Silva LS, Scarabelot VL, Ströher R, de Souza A, Fregni F, Pereira P. Transcranial direct current stimulation (tDCS) affects neuroinflammation parameters and behavioral seizure activity in pentylenetetrazole-induced kindling in rats. Neurosci Lett. 2020 Sep 14;735:135162.