Beneficial effects of L-arginine on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neuronal degeneration in substantia nigra of Balb/c mice


1 Department of Anatomical Sciences, School of Medicine, Birjand University of Medical Sciences, Birjand, Iran

2 Department of Public Health, Research Centre of Experimental Medicine, Deputy of Research and Technology, Birjand University of Medical Sciences, Birjand, Iran

3 Department of Biology, School of Sciences, Payam-e-Noor University, Tehran, Iran

4 Student of Medicine, Department of Anatomical Sciences, School of Medicine, Birjand University of Medical Sciences, Birjand, Iran

5 Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Background: L-arginine has been recently investigated and proposed to reduce neurological damage after various experimental models of neuronal cellular damage. In this study, we aim to evaluate the beneficial effects of L-arginine administration on the numerical density of dark neurons (DNs) in the substantia nigra pars compacta (SNc) of Balb/c mice subjected to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration.
Materials and Methods: Male Balb/c mice were randomly divided into 4 groups (n = 7 each): MPTP only; saline only (control); MPTP + L-arginine; and L-arginine only. The animals were infused intranasally with a single intranasal administration of the proneurotoxin MPTP (1 mg/nostril). L-arginine (300 mg/kg) was administrated intraperitoneally once daily for 1-week starting from 3 days after MPTP administration. Cavalieri principle method was used to estimate the numerical density of DNs in the SNc of different studied groups.
Results: Twenty days following MPTP administration, the number of DNs was significantly increased when compared to sham-control and L-arginine-control groups (P < 0.05). Nevertheless, our results showed that L-arginine administration significantly decreased the numerical density of DNs in SNc of mice.
Conclusion: This investigation provides new insights in experimental models of Parkinson's disease, indicating that L-arginine represents a potential treatment agent for dopaminergic neuron degeneration in SNc observed in Parkinson's disease patients.


Schapira AH, Jenner P. Etiology and pathogenesis of Parkinson's disease. Mov Disord 2011;26:1049-55.  Back to cited text no. 1
Thenganatt MA, Jankovic J. Parkinson disease subtypes. JAMA Neurol 2014;71:499-504.  Back to cited text no. 2
Alves G, Forsaa EB, Pedersen KF, Dreetz Gjerstad M, Larsen JP. Epidemiology of Parkinson's disease. J Neurol 2008;255 Suppl 5:18-32.  Back to cited text no. 3
Shulman JM, De Jager PL, Feany MB. Parkinson's disease: Genetics and pathogenesis. Annu Rev Pathol 2011;6:193-222.  Back to cited text no. 4
Li LH, Qin HZ, Wang JL, Wang J, Wang XL, Gao GD. Axonal degeneration of nigra-striatum dopaminergic neurons induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. J Int Med Res 2009;37:455-63.  Back to cited text no. 5
Herrero MT, Luquín MR, Obeso JA. Experimental model of Parkinson disease: Mechanisms and anatomo- pathological characteristics of MPTP neurotoxicity. Arch Neurobiol (Madr) 1992;55:175-82.  Back to cited text no. 6
Kopin IJ. Features of the dopaminergic neurotoxin MPTP. Ann N Y Acad Sci 1992;648:96-104.  Back to cited text no. 7
Lessel J. MPTP – Neurotoxin and model substance in Parkinson research. Pharm Unserer Zeit 1994;23:106-7.  Back to cited text no. 8
Smeyne RJ, Jackson-Lewis V. The MPTP model of Parkinson's disease. Brain Res Mol Brain Res 2005;134:57-66.  Back to cited text no. 9
He XJ, Nakayama H, Dong M, Yamauchi H, Ueno M, Uetsuka K, et al. Evidence of apoptosis in the subventricular zone and rostral migratory stream in the MPTP mouse model of Parkinson disease. J Neuropathol Exp Neurol 2006;65:873-82.  Back to cited text no. 10
Prediger RD, Aguiar AS Jr, Rojas-Mayorquin AE, Figueiredo CP, Matheus FC, Ginestet L, et al. Single intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57BL/6 mice models early preclinical phase of Parkinson's disease. Neurotox Res 2010;17:114-29.  Back to cited text no. 11
Roy A, Ghosh A, Jana A, Liu X, Brahmachari S, Gendelman HE, et al. Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopaminergic neurons in mouse models of Parkinson's disease. PLoS One 2012;7:e38113.  Back to cited text no. 12
Dluzen DE, Kefalas G. The effects of intranasal infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon catecholamine concentrations within olfactory bulbs and corpus striatum of male mice. Brain Res 1996;741:215-9.  Back to cited text no. 13
Prediger RD, Aguiar AS Jr, Moreira EL, Matheus FC, Castro AA, Walz R, et al. The intranasal administration of 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP): A new rodent model to test palliative and neuroprotective agents for Parkinson's disease. Curr Pharm Des 2011;17:489-507.  Back to cited text no. 14
Prediger RD, Batista LC, Medeiros R, Pandolfo P, Florio JC, Takahashi RN. The risk is in the air: Intranasal administration of MPTP to rats reproducing clinical features of Parkinson's disease. Exp Neurol 2006;202:391-403.  Back to cited text no. 15
Prediger RD, Rial D, Medeiros R, Figueiredo CP, Doty RL, Takahashi RN. Risk is in the air: An intranasal MPTP (1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine) rat model of Parkinson's disease. Ann N Y Acad Sci 2009;1170:629-36.  Back to cited text no. 16
Ransom BR, Kunis DM, Irwin I, Langston JW. Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP. Neurosci Lett 1987;75:323-8.  Back to cited text no. 17
Cui M, Aras R, Christian WV, Rappold PM, Hatwar M, Panza J, et al. The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway. Proc Natl Acad Sci U S A 2009;106:8043-8.  Back to cited text no. 18
Jenner P. Oxidative stress as a cause of Parkinson's disease. Acta Neurol Scand Suppl 1991;136:6-15.  Back to cited text no. 19
Jackson-Lewis V, Jakowec M, Burke RE, Przedborski S. Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurodegeneration 1995;4:257-69.  Back to cited text no. 20
Virarkar M, Alappat L, Bradford PG, Awad AB. L-arginine and nitric oxide in CNS function and neurodegenerative diseases. Crit Rev Food Sci Nutr 2013;53:1157-67.  Back to cited text no. 21
Yi J, Horky LL, Friedlich AL, Shi Y, Rogers JT, Huang X. L-arginine and Alzheimer's disease. Int J Clin Exp Pathol 2009;2:211-38.  Back to cited text no. 22
Rassaf T, Kleinbongard P, Kelm M. The L-arginine nitric oxide pathway: Avenue for a multiple-level approach to assess vascular function. Biol Chem 2006;387:1347-9.  Back to cited text no. 23
Kelm M. The L-arginine-nitric oxide pathway in hypertension. Curr Hypertens Rep 2003;5:80-6.  Back to cited text no. 24
Olken NM, Osawa Y, Marletta MA. Characterization of the inactivation of nitric oxide synthase by NG-methyl-L-arginine: Evidence for heme loss. Biochemistry 1994;33:14784-91.  Back to cited text no. 25
Barthwal MK, Srivastava N, Dikshit M. Role of nitric oxide in a progressive neurodegeneration model of Parkinson's disease in the rat. Redox Rep 2001;6:297-302.  Back to cited text no. 26
Santos RM, Lourenço CF, Ledo A, Barbosa RM, Laranjinha J. Nitric oxide inactivation mechanisms in the brain: Role in bioenergetics and neurodegeneration. Int J Cell Biol 2012;2012:391914.  Back to cited text no. 27
Contestabile A, Monti B, Contestabile A, Ciani E. Brain nitric oxide and its dual role in neurodegeneration/neuroprotection: Understanding molecular mechanisms to devise drug approaches. Curr Med Chem 2003;10:2147-74.  Back to cited text no. 28
Molina JA, Jiménez-Jiménez FJ, Ortí-Pareja M, Navarro JA. The role of nitric oxide in neurodegeneration. Potential for pharmacological intervention. Drugs Aging 1998;12:251-9.  Back to cited text no. 29
Youdim MB, Lavie L, Riederer P. Oxygen free radicals and neurodegeneration in Parkinson's disease: A role for nitric oxide. Ann N Y Acad Sci 1994;738:64-8.  Back to cited text no. 30
Lundblad C, Bentzer P. Effects of L-arginine on cerebral blood flow, microvascular permeability, number of perfused capillaries, and brain water content in the traumatized mouse brain. Microvasc Res 2007;74:1-8.  Back to cited text no. 31
Willmot M, Gray L, Gibson C, Murphy S, Bath PM. A systematic review of nitric oxide donors and L-arginine in experimental stroke; effects on infarct size and cerebral blood flow. Nitric Oxide 2005;12:141-9.  Back to cited text no. 32
Kovách AG, Szabó C, Benyó Z, Csáki C, Greenberg JH, Reivich M. Effects of NG-nitro-L-arginine and L-arginine on regional cerebral blood flow in the cat. J Physiol 1992;449:183-96.  Back to cited text no. 33
Ishida K, Shimizu H, Hida H, Urakawa S, Ida K, Nishino H. Argyrophilic dark neurons represent various states of neuronal damage in brain insults: Some come to die and others survive. Neuroscience 2004;125:633-44.  Back to cited text no. 34
Gallyas F, Kiglics V, Baracskay P, Juhász G, Czurkó A. The mode of death of epilepsy-induced “dark” neurons is neither necrosis nor apoptosis: An electron-microscopic study. Brain Res 2008;1239:207-15.  Back to cited text no. 35
Kherani ZS, Auer RN. Pharmacologic analysis of the mechanism of dark neuron production in cerebral cortex. Acta Neuropathol 2008;116:447-52.  Back to cited text no. 36
Ahmadpour SH, Haghir H. Diabetes mellitus type 1 induces dark neuron formation in the dentate gyrus: A study by Gallyas' method and transmission electron microscopy. Rom J Morphol Embryol 2011;52:575-9.  Back to cited text no. 37
Cammermeyer J. I. An evaluation of the significance of the “dark” neuron. Ergeb Anat Entwicklungsgesch 1962;36:1-61.  Back to cited text no. 38
Garman RH. The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation. Neurotoxicology 2006;27:1126.  Back to cited text no. 39
Jortner BS. The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation. Neurotoxicology 2006;27:628-34.  Back to cited text no. 40
Jafarian M, Rahimi S, Behnam F, Hosseini M, Haghir H, Sadeghzadeh B, et al. The effect of repetitive spreading depression on neuronal damage in juvenile rat brain. Neuroscience 2010;169:388-94.  Back to cited text no. 41
Pucaj K, Rasmussen H, Møller M, Preston T. Safety and toxicological evaluation of a synthetic vitamin K2, menaquinone-7. Toxicol Mech Methods 2011;21:520-32.  Back to cited text no. 42
Matheus FC, Aguiar AS Jr, Castro AA, Villarinho JG, Ferreira J, Figueiredo CP, et al. Neuroprotective effects of agmatine in mice infused with a single intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Behav Brain Res 2012;235:263-72.  Back to cited text no. 43
Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 6th ed. New York: Elsevier; 2006.  Back to cited text no. 44
Gundersen HJ, Bagger P, Bendtsen TF, Evans SM, Korbo L, Marcussen N, et al. The new stereological tools: Disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 1988;96:857-81.  Back to cited text no. 45
Meissner W, Hill MP, Tison F, Gross CE, Bezard E. Neuroprotective strategies for Parkinson's disease: Conceptual limits of animal models and clinical trials. Trends Pharmacol Sci 2004;25:249-53.  Back to cited text no. 46
Cherian L, Chacko G, Goodman C, Robertson CS. Neuroprotective effects of L-arginine administration after cortical impact injury in rats: Dose response and time window. J Pharmacol Exp Ther 2003;304:617-23.  Back to cited text no. 47
Rosa AO, Lin J, Calixto JB, Santos AR, Rodrigues AL. Involvement of NMDA receptors and L-arginine-nitric oxide pathway in the antidepressant-like effects of zinc in mice. Behav Brain Res 2003;144:87-93.  Back to cited text no. 48
Freitas AE, Moretti M, Budni J, Balen GO, Fernandes SC, Veronezi PO, et al. NMDA receptors and the L-arginine-nitric oxide-cyclic guanosine monophosphate pathway are implicated in the antidepressant-like action of the ethanolic extract from Tabebuia avellanedae in mice. J Med Food 2013;16:1030-8.  Back to cited text no. 49
Ates-Alagoz Z, Adejare A. NMDA Receptor Antagonists for Treatment of Depression. Pharmaceuticals (Basel) 2013;6:480-99.  Back to cited text no. 50
Jadeski LC, Lala PK. Nitric oxide synthase inhibition by N (G)-nitro-L-arginine methyl ester inhibits tumor-induced angiogenesis in mammary tumors. Am J Pathol 1999;155:1381-90.  Back to cited text no. 51
Tripathi P, Misra MK. Therapeutic role of L-arginine on free radical scavenging system in ischemic heart diseases. Indian J Biochem Biophys 2009;46:498-502.  Back to cited text no. 52
Dedkova EN, Blatter LA. Characteristics and function of cardiac mitochondrial nitric oxide synthase. J Physiol 2009;587:851-72.  Back to cited text no. 53
Lerman A, Burnett JC Jr, Higano ST, McKinley LJ, Holmes DR Jr. Long-term L-arginine supplementation improves small-vessel coronary endothelial function in humans. Circulation 1998;97:2123-8.  Back to cited text no. 54
Buchanan JE, Phillis JW. The role of nitric oxide in the regulation of cerebral blood flow. Brain Res 1993;610:248-55.  Back to cited text no. 55
Garry PS, Ezra M, Rowland MJ, Westbrook J, Pattinson KT. The role of the nitric oxide pathway in brain injury and its treatment – From bench to bedside. Exp Neurol 2015;263:235-43.  Back to cited text no. 56
Garthwaite J, Boulton CL. Nitric oxide signaling in the central nervous system. Annu Rev Physiol 1995;57:683-706.  Back to cited text no. 57
Iadecola C. Regulation of the cerebral microcirculation during neural activity: Is nitric oxide the missing link? Trends Neurosci 1993;16:206-14.  Back to cited text no. 58
Blandini F, Greenamyre JT, Nappi G. The role of glutamate in the pathophysiology of Parkinson's disease. Funct Neurol 1996;11:3-15.  Back to cited text no. 59
Hazell AS, Itzhak Y, Liu H, Norenberg MD. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) decreases glutamate uptake in cultured astrocytes. J Neurochem 1997;68:2216-9.  Back to cited text no. 60
Cherian L, Chacko G, Goodman JC, Robertson CS. Cerebral hemodynamic effects of phenylephrine and L-arginine after cortical impact injury. Crit Care Med 1999;27:2512-7.  Back to cited text no. 61
Condello S, Calabrò E, Caccamo D, Currò M, Ferlazzo N, Satriano J, et al. Protective effects of agmatine in rotenone-induced damage of human SH-SY5Y neuroblastoma cells: Fourier transform infrared spectroscopy analysis in a model of Parkinson's disease. Amino Acids 2012;42:775-81.  Back to cited text no. 62
Martínez-Orgado J, Fernández-Frutos B, González R, Fernández-López D, Urigüen L, Romero E, et al. Neuroprotective effect of L-arginine in a newborn rat model of acute severe asphyxia. Biol Neonate 2005;88:291-8.  Back to cited text no. 63