B Vitamins Supplement Potentiates Antiparkinsonian Effect of Flunarizine: the Behavioral and Biochemical Evidences From 6-Hydroxydopamine Animal Model

Document Type: Original Article


1 Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

2 Associate Professor, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran


Introduction: Prominent data indicate that flunarizine (flu), a calcium channel blocker, has neuroprotective effect. However, several authors have reported that the chronic use of flu can produce drug-induced Parkinsonism. Previously, we showed that B vitamins supplement (B com) has antiparkinsonian effect. In the present study, we evaluated the effect of pretreatment with flu and a combination of flu and B com on the 6-hydroxydopamine (6-OHDA) - induced Parkinsonism.
Methods: 6-OHDA (4 μl, 4 μg/μl) was injected into right striatum by stereotaxic surgery. Different groups of rats received flu (5 or 10 mg/kg) or B com or a combination of them before the toxin to three weeks after that. The severity of Parkinsonism was assessed by conventional behavioral tests and also biochemical measurement of striatal dopamine level. Furthermore, malondialdehyde (MDA) concentration was measured in the serum and brain suspension.
Results: Pretreatments with flu or B com significantly attenuated apomorphine- induced rotations and improved rotarod performance, but they had little effect on the 6- OHDA- induced swinging behavior. The pretreatments also reduced the decreasing effect of 6- OHDA on the striatal dopamine level. These antiparkinsonian effects were potentiated when animals were pretreated with a combination of flu and B com. In addition, B com alone or in combination with flu reduced MDA concentration especially in the brain tissue. On the other hand, flu increased MDA concentration in the serum.
Conclusion: Our data show that co-administration of B com with flu potentiate largely the antiparkinsonian effect and may attenuate its adverse effects.


  1. Hancock DB, Martin ER, Stajich JM, Jewett R, Stacy MA, Scott BL, et al. Smoking, caffeine, and nonsteroidal anti-inflammatory drugs in families with Parkinson disease. Arch Neurol 2007; 64:576-80.
  2. Jenner P, Olanow CW. Oxidative stress and the pathogenesis of parkinson’s disease. Neurology 1996; 47(6 Suppl 3):161-70.
  3. de Lau LM, Koudstaal PJ, Van Meurs JB, Uitterlinder AG, Hofman A, Breteler MM. Methylenetetrahydrofolate reductase C677T genotype and PD. Ann Neurol 2005; 57(6):927-30.
  4. Tatton NA. Increased caspase 3 and bax immunoreactivity accompany nuclear GAPDH translocation and neuronal apoptosis in parkinson’s disease. Exp Neurol 2000; 166(1):29-43.
  5. Cano Abad MF, Villarroya M, García AG, L´opez MG. Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death. J Biol Chem 2001; 276(43):39695-704.
  6. Erami E, Azhdari Zarmehri H, Ghasemi Dashkhasan E, Esmaeili MH, Semnanian S. Intra-paragigantocellularis lateralis injection of orexin-A has an antinociceptive effect on hot plate and formalin tests in rat.Brain Res2012; 1478:16-23.
  7. Surmeier DJ, Schumacker PT. Calcium, bioenergetics, and neuronal vulnerability in Parkinson’s disease. J Biol Chem 2013; 288(15):10736-41.
  8. Williams ME, Brust PF, Feldman DH, Patthi S, Simerson S, Maroufi A, et al. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science 1992; 257(5068):389-95.
  9. Díaz-Prieto N, Herrera-Peco I, de Diego AM, Ruiz-Nuño A, Gallego-Sandín S, López MG, et al. Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through down regulation of L-type Ca2+ channels in PC12 cells. Cell Calcium 2008; 44(4):339-52.
  10. Ilijic E, Guzman JN, Surmeier DJ. The L-type channel antagonist isradipine is neuroprotective in a mouse model of Parkinson’s disease. Neurobiol Dis 2011; 43(2):364-71.
  11. Qu L, Wang Y, Zhang HT, Li N, Wang Q, Yang Q, et al. 6-OHDA induced calcium influx through N-type calcium channel alters membrane properties via PKA pathway in substantia nigra pars compacta dopaminergic neurons. Neurosci Lett 2014; 575:1-6.
  12. Haghdoost Yazdi H, Hosseini SS, Faraji A, Nahid D, Jahanihashemi H. Long term exposure to norharman exacerbates 6-hydroxydopamine-induced parkinsonism: possible involvement of L-type Ca2+ channels. Behav Brain Res 2010; 215(1):136-40.
  13. Annoura H, Nakanishi K, Uesugi M, Fukunaga A, Imajo S, Miyajima A, et al. Synthesis and biological evaluation of new 4-arylpiperidines and 4-Aryl-4-piperidinols: dual Na+ and Ca2+ channel blockers with reduced affinity for dopamine D2 receptors. Bioorganic & Medicinal Chemistry 2002; 10(2):371-83.
  14. Pauwels PJ, Leysen JE, Janssen PA. Ca++ and Na+ channels involved in neuronal cell death. Protection by flunarizine. Life Sci 1991; 48(20):1881-93.
  15. Van Zwieten PA. Calcium antagonists, calcium entry blockers and calcium overload blockers; nomenclature and classification. Ned Tijdschr Geneeskd 1985; 129(17):777-80 [In Dutch].
  16. Rich KM, Hollowell JP. Flunarizine protects neurons from death after axotomy or NGF deprivation. Science 1990; 248(4961):1419-21.
  17. Maroto R, De la Fuente MT, Artalejo AR, Abad F, López MG, García-Sancho J, et al. Effects of Ca2+ channel antagonists on chromaffin cell death and cytosolic Ca2+ oscillations induced by veratridine. Eur J Pharmacol 1994; 270(4):331-9.
  18. Ashton D, Willems R, Marrannes R, Janssen PA. Extracellular ions during veratridine-induced neurotoxicity in hippocampal slices: neuroprotective effects of flunarizine and tetrodotoxin. Brain Res 1990; 528(2):212-22.
  19. De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen PA. Photochemical stroke model: flunarizine prevents sensorimotor deficits after neocortical infarcts in rats. Stroke 1989; 20(10):1383-90.
  20. Brücke T, Wöber CH, Podreka I, Wöber Bingöl C, Asenbaum S, Aull S, et al. D2 receptor blockade by flunarizine and cinnarizine explains extrapyramidal side effects. J Cereb Blood Flow Metab 1995; 15(3):513-8.
  21. Fabiani G, Pastro PC, Froehner C. Parkinsonism and other movement disorders in outpatients in chronic use of cinnarizine and flunarizine. Arq Neuropsiquiatr 2004; 62(3B):784-88.
  22. Teive HA, Troiano AR, Germiniani FM, Werneck LC. Flunarizine and cinnarizine-induced parkinsonism: a historical and clinical analysis. Parkinsonism Relat Disord 2004; 10(4):243-45.
  23. Mena MA, Garcia de Yébenes MJ, Tabernero C, Casarejos MJ, Pardo B, Garcia de Yébenes J. Effects of calcium antagonists on the dopamine system. Clin Neuropharmacol 1995; 18(5):410-26.
  24. Fraidouni N, Sarookhani M, Sophiabadi M, Haghdoost Yazdi H. High intake of folic acid attenuates 6-hydroxydopamine-induced parkinsonism in rats independent of serum level of homocysteine. Physiology and Pharmacology 2012; 16(3):231-44.
  25. Haghdoost Yazdi H, Fraidouni N, Faraji A, Jahanihashemi H, Sarookhani M. High intake of folic acid or complex of B vitamins provides anti-Parkinsonism effect: no role for serum level of homocysteine. Behav Brain Res 2012; 233(2):375-81.
  26. Sophiabadi M, Fraidouni N, Faraji A, Dargahi T, Yaghubi Dust H,  Haghdoost Yazdi H. Effect of tetraethylammonium and B vitamins group on the efficacy of cell replacement therapy in the treatment of parkinson's disease in the 6-hydroxydopamine animal model. Physiology and Pharmacology 2013; 17(3):266-76. [In Persian].
  27. Iancu R, Mohapel P, Brundin P, Paul G. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson's disease in mice. Behav Brain Res 2005; 162(1):1-10.
  28. Shimohama S, Sawada H, Kitamura Y, Taniguchi T. Disease model: parkinson's disease. Trends Mol Med 2003; 9(8):360-5.
  29. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 6th ed. San Diego: Academic Press; 2007.
  30. Borlongan CV, Sanberg PR. Elevated body swing test: a new behavioral parameter for rats with 6-hydroxydopamine-induced hemiparkinsonism. J Neurosci 1995; 15(7 Pt 2):5372-8.
  31. Albro PW, Corbett JT, Schroeder JL. Application of the thiobarbiturate assay to the measurement of lipid peroxidation products in microsomes. J Biochem Biophys Methods 1986; 13(3):185-94.
  32. Borlongan CV, Randall TS, Cahill DW, Sanberg PR. Asymmetrical motor behavior in rats with unilateral striatal excitotoxic lesions as revealed by the elevated body swing test. Brain Res 1995; 676(1):231-4.
  33. Sarookhani MR, Haghdoost Yazdi H, Sarbazi Golezari A, Babayan Tazehkand A, Rastgoo N. Involvement of adenosine triphosphate-sensitive potassium channels in the neuroprotective activity of hydrogen sulfide in the 6-hydroxydopamine-induced animal model of Parkinson's disease. Behav Pharmacol 2018; 29(4):336-43.
  34. Sarukhani MR, Haghdoost Yazdi H, Khandan Chelarci G. changes in the serum urate level can predict the development of parkinsonism in the 6-hydroxydopamine animal model. Neurochem Res 2018; 43(5):1086-95.
  35. Yuan H, Sarre S, Ebinger G, Michotte Y. Histological, behavioral and neurochemical evaluation of medial forebrain bundle and striatal 6-OHDA lesions as rat models of Parkinson's disease. J Neurosci Methods 2005; 144(1):35-45.
  36. Dauer W, Przedborskim S. Parkinson's disease: mechanisms and models. Neuron 2003; 39(6):889-909.
  37. Abrous DN, Rodriguez JJ, Montaron MF, Aurousseau C, Le Moal M, Barneoud P. Behavioural recovery after unilateral lesion of the dopaminergic mesotelencephalic pathway: effect of repeated testing. Neuroscience 1998; 84(1):213-21.
  38. Haghdoost Yazdi H, Sarookhani M, Faraj A, Fraidouni N, Dargahi T, Yaghoubidoust MH, et al. Evaluation of the association between blood homocysteine concentration and the degree of behavioral symptoms in the 6-hydroxydopamine-induced Parkinsonism in rat. Pharmacol Biochem Behav 2014; 124:297-304.
  39. Chen TF, Chiu MJ, Huang CT, Tang MC, Wang SJ, Wang CC, et al. Changes in dietary folate intake differentially affect oxidized lipid and mitochondrial DNA damage in various brain regions of rats in the absence/presence of intracerebroventricularly injected amyloid b-peptide challenge. Br J Nutr 2011; 105(9):1294-302.
  40. Jia H, Liu Z, Li X, Feng Z, Hao J, Li X, et al. Synergistic anti-Parkinsonism activity of high doses of B vitamins in a chronic cellular model. Neurobiol Aging 2010; 31(4):636-46.
  41. Patro IK, Chattopadhyay M, Patro N. Flunarizine enhances functional recovery following sciatic nerve crush lesion in rats. Neurosci Lett 1999; 263(2-3):97-100.
  42. Kaminski Schierle GS, Hansson O, Brundin P. Flunarizine improves the survival of grafted dopaminergic neurons. Neuroscience 1999; 94(1):17-20.
  43. Scheufler E, Peters T. Phosphatidylserine monolayers as models for drug uptake into membranes and tissue. Cell Biol Int Rep 1990; 14(4):381-8.
  44. Thomas PG, Seelig J. Binding of the calcium antagonist flunarizine to phosphatidylcholine bilayers: charge effects and thermodynamics. Biochem J 1993; 291(Pt 2):397-402.
  45. Maroto R, López MG, del Valle M, Naranjo JR, Mellström B, García AG. Expression of the bovine striatal D2 receptor, but not the D1 receptor, in bovine adrenal medulla. Mol. Pharmacol 1995; 47(1):40-50.
  46. Novalbos J, Abad-Santos F, Zapater P, Cano-Abad MF, Moradiellos J, Sánchez-García P, et al. Effects of dotarizine and flunarizine on chromaffin cell viability and cytosolic Ca2+. Eur J Pharmacol 1999; 366(2-3):309-17.
  47. Elimadi A, Bouillot L, Sapena R, Tillement JP, Morin D. Dose-related inversion of cinnarizine and flunarizine effects on mitochondrial permeability transition. Eur J Pharmacol 1998; 348(1):115-21.
  48. Cruz TS, Faria PA, Santana DP, Ferreira JC, Oliveira V, Nascimento OR, et al. Nantes IL and Rodrigues T. On the mechanisms of phenothiazine-induced mitochondrial permeability transition: thiol oxidation, strict Ca2+ dependence, and cyt c release. Biochem Pharmacol 2010; 80(8):1284-95.
  49. Silverstein FS, Buchanan K, Hudson C, Johnston MV. Flunarizine limits hypoxia-ischemia induced morphologic injury in immature rat brain. Stroke1986; 17(3):477-82.
  50. Gunn AJ, Gluckman PD. Flunarizine, a calcium channel antagonist, is not neuroprotective when given after hypoxia-ischemia in the infant rat. Dev Pharmacol Ther 1991; 17(3-4):205-9.
  51. Gunn AJ, Williams CE, Mallard EC, Tan WK, Gluckman PD. Flunarizine, a calcium channel antagonist, is partially prophylactically neuroprotective in hypoxic-ischemic encephalopathy in the fetal sheep. Pediatr Res 1994; 35(6):657-63.
  52. Gunn AJ, Gluckman PD. Flunarizine, a calcium channel antagonist, is not neuroprotective when given after hypoxia-ischemia in the infant rat. Dev Pharmacol Ther 1991; 17(3-4):205-9.
  53. Gunn AJ, Williams CE, Mallard EC, Tan WK, Gluckman PD. Flunarizine, a calcium channel antagonist, is partially prophylactically neuroprotective in hypoxic-ischemic encephalopathy in the fetal sheep. Pediatr Res 1994; 35(6):657-63.