Neurotoxicity Research

, Volume 15, Issue 3, pp 260–273

Methylene Blue Provides Behavioral and Metabolic Neuroprotection Against Optic Neuropathy

  • Julio C. Rojas
  • Joseph M. John
  • Jung Lee
  • F. Gonzalez-Lima
Article

Abstract

Methylene blue (MB) is a diaminophenothiazine with potent antioxidant and unique redox properties that prevent morphologic degenerative changes in the mouse retina induced by rotenone, a specific mitochondrial complex I inhibitor. This study evaluated pigmented rats to determine whether MB’s neuroprotective effects against rotenone-mediated retinal neurotoxicity have functional relevance and whether these effects are mediated by an improvement in neuronal energy metabolism in vivo. Visual function was behaviorally assessed by determining differences in the illuminance sensitivity threshold pre- and post-bilateral intravitreal injection of rotenone (200 μg/kg) or rotenone plus MB (70 μg/kg). Retinal degeneration was morphologically studied using unbiased stereological tools. Changes in histochemically determined cytochrome oxidase activity in the visual pathway were used to evaluate the impact of treatments on neuronal energy metabolism. Rotenone induced a 1.4 log unit increase in the illumination threshold compared to baseline, as well as a 32% decrease in ganglion cell layer cell (GCL) density, and a 56% decrease in GCL layer + nerve fiber layer thickness. Co-administration of MB prevented the changes in visual function and the retinal histopathology. Furthermore, rotenone induced a functional deafferentation of the visual system, as revealed by decreases in the metabolic activity of the retina, superior colliculus, and visual cortex. These metabolic changes were also prevented by MB. The results provided the first demonstration of MB’s behavioral and metabolic neuroprotection against optic neuropathy, and implicate MB as a candidate neuroprotective agent with metabolic-enhancing properties that may be used in the treatment of neurodegenerative diseases associated with mitochondrial dysfunction.

Keywords

Methylene blue Rotenone Cytochrome oxidase Neuroprotection Neurodegeneration Retinal neurotoxicity 

References

  1. Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, Kato H, Ames BN (2008) Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J 22:703–712PubMedCrossRefGoogle Scholar
  2. Beretta S, Wood JP, Derham B, Sala G, Tremolizzo L, Ferrarese C, Osborne NN (2006) Partial mitochondrial complex I inhibition induces oxidative damage and perturbs glutamate transport in primary retinal cultures. Relevance to Leber Hereditary Optic Neuropathy (LHON). Neurobiol Dis 24:308–317PubMedCrossRefGoogle Scholar
  3. Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT (2000) Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci 3:1301–1306PubMedCrossRefGoogle Scholar
  4. Borit A (1971) Leigh’s necrotizing encephalomyelopathy, Neuro-ophthalmological abnormalities. Arch Ophthalmol 85:438–442PubMedGoogle Scholar
  5. Brown MD, Zhadanov S, Allen JC, Hosseini S, Newman NJ, Atamonov VV, Mikhailovskaya IE, Sukernik RI, Wallace DC (2001) Novel mtDNA mutations and oxidative phosphorylation dysfunction in Russian LHON families. Hum Genet 109:33–39PubMedCrossRefGoogle Scholar
  6. Bruchey AK, Gonzalez-Lima F (2006) Brain activity associated with fear renewal. Eur J Neurosci 24:3567–3577PubMedCrossRefGoogle Scholar
  7. Buchholz K, Schirmer RH, Eubel JK, Akoachere MB, Dandekar T, Becker K, Gromer S (2008) Interactions of methylene blue with human disulfide reductases and their orthologues from Plasmodium falciparum. Antimicrob Agents Chemother 52:183–191PubMedCrossRefGoogle Scholar
  8. Callaway NL, Riha PD, Wrubel KM, McCollum D, Gonzalez-Lima F (2002) Methylene blue restores spatial memory retention impaired by an inhibitor of cytochrome oxidase in rats. Neurosci Lett 332:83–86PubMedCrossRefGoogle Scholar
  9. Callaway NL, Riha PD, Bruchey AK, Munshi Z, Gonzalez-Lima F (2004) Methylene blue improves brain oxidative metabolism and memory retention in rats. Pharmacol Biochem Behav 77:175–181PubMedCrossRefGoogle Scholar
  10. Carelli V, Ross-Cisneros FN, Sadun AA (2002) Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies. Neurochem Int 40:573–584PubMedCrossRefGoogle Scholar
  11. Casari G, De Fusco M, Ciarmatori S, Zeviani M, Mora M, Fernandez P, De Michele G, Filla A, Cocozza S, Marconi R, Durr A, Fontaine B, Ballabio A (1998) Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 93:973–983PubMedCrossRefGoogle Scholar
  12. Chalmers RM, Schapira AH (1999) Clinical, biochemical and molecular genetic features of Leber’s hereditary optic neuropathy. Biochim Biophys Acta 1410:147–158PubMedCrossRefGoogle Scholar
  13. Chinnery PF, Howell N, Lightowlers RN, Turnbull DM (1997) Molecular pathology of MELAS and MERRF. The relationship between mutation load and clinical phenotypes. Brain 120(Pt 10):1713–1721PubMedCrossRefGoogle Scholar
  14. Clark JM, Switzer RL (1977) Experimental biochemistry, 2nd edn. W.H. Freeman and Company, San FranciscoGoogle Scholar
  15. Danesh-Meyer HV, Birch H, Ku JY, Carroll S, Gamble G (2006) Reduction of optic nerve fibers in patients with Alzheimer disease identified by laser imaging. Neurology 67:1852–1854PubMedCrossRefGoogle Scholar
  16. Degli Esposti M (1998) Inhibitors of NADH-ubiquinone reductase: an overview. Biochim Biophys Acta 1364:222–235PubMedCrossRefGoogle Scholar
  17. Degli Esposti M, Lenaz G (1982) Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles. Biochim Biophys Acta 682:189–200PubMedCrossRefGoogle Scholar
  18. DiMauro S (1999) Mitochondrial encephalomyopathies: back to Mendelian genetics. Ann Neurol 45:693–694PubMedCrossRefGoogle Scholar
  19. Drew B, Leeuwenburgh C (2003) Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. Am J Physiol Regul Integr Comp Physiol 285:R1259–R1267PubMedGoogle Scholar
  20. Eells JT, Henry MM, Summerfelt P, Wong-Riley MT, Buchmann EV, Kane M, Whelan NT, Whelan HT (2003) Therapeutic photobiomodulation for methanol-induced retinal toxicity. Proc Natl Acad Sci USA 100:3439–3444PubMedCrossRefGoogle Scholar
  21. Estornell E, Fato R, Pallotti F, Lenaz G (1993) Assay conditions for the mitochondrial NADH: coenzyme Q oxidoreductase. FEBS Lett 332:127–131PubMedCrossRefGoogle Scholar
  22. Galili Y, Ben-Abraham R, Weinbroum A, Marmur S, Iaina A, Volman Y, Peer G, Szold O, Soffer D, Klausner J, Rabau M, Kluger Y (1998) Methylene blue prevents pulmonary injury after intestinal ischemia-reperfusion. J Trauma 45:222–225; discussion 225–226PubMedCrossRefGoogle Scholar
  23. Gonzalez-Lima F, Bruchey AK (2004) Extinction memory improvement by the metabolic enhancer methylene blue. Learn Mem 11:633–640PubMedCrossRefGoogle Scholar
  24. Gonzalez-Lima F, Cada A (1994) Cytochrome oxidase activity in the auditory system of the mouse: a qualitative and quantitative histochemical study. Neuroscience 63:559–578PubMedCrossRefGoogle Scholar
  25. Gonzalez-Lima F, Cada A (1998) Quantitative histochemistry of cytochrome oxidase activity: theory, methods, and regional brain vulnerability. In: Gonzalez-Lima F (ed) Cytochrome oxidase in neuronal metabolism, Alzheimer’s disease. Plenum press, New York, pp 55–90Google Scholar
  26. Gonzalez-Lima F, Jones D (1994) Quantitative mapping of cytochrome oxidase activity in the central auditory system of the gerbil: a study with calibrated activity standards and metal-intensified histochemistry. Brain Res 660:34–49PubMedCrossRefGoogle Scholar
  27. Gundersen HJ, Jensen TB, Osterby R (1978) Distribution of membrane thickness determined by lineal analysis. J Microsc 113:27–43PubMedGoogle Scholar
  28. Harding AJ, Halliday GM, Cullen K (1994) Practical considerations for the use of the optical disector in estimating neuronal number. J Neurosci Methods 51:83–89PubMedCrossRefGoogle Scholar
  29. Hayes JM, Balkema GW (1993) Elevated dark-adapted thresholds in hypopigmented mice measured with a water maze screening apparatus. Behav Genet 23:395–403PubMedCrossRefGoogle Scholar
  30. Hevner RF, Wong-Riley MT (1990) Regulation of cytochrome oxidase protein levels by functional activity in the macaque monkey visual system. J Neurosci 10:1331–1340PubMedGoogle Scholar
  31. Hinton DR, Sadun AA, Blanks JC, Miller CA (1986) Optic-nerve degeneration in Alzheimer’s disease. N Engl J Med 315:485–487PubMedCrossRefGoogle Scholar
  32. Hu D, Xu X, Gonzalez-Lima F (2006) Vicarious trial-and-error behavior and hippocampal cytochrome oxidase activity during Y-maze discrimination learning in the rat. Int J Neurosci 116:265–280PubMedCrossRefGoogle Scholar
  33. Hwang JM, Park HW, Kim SJ (1997) Optic neuropathy associated with mitochondrial tRNA[Leu(UUR)] A3243G mutation. Ophthalmic Genet 18:101–105PubMedCrossRefGoogle Scholar
  34. Ikegami K, Koike T (2003) Non-apoptotic neurite degeneration in apoptotic neuronal death: pivotal role of mitochondrial function in neurites. Neuroscience 122:617–626PubMedCrossRefGoogle Scholar
  35. Iseri PK, Altinas O, Tokay T, Yuksel N (2006) Relationship between cognitive impairment and retinal morphological and visual functional abnormalities in Alzheimer disease. J Neuroophthalmol 26:18–24PubMedGoogle Scholar
  36. Jung C, Higgins CM, Xu Z (2002) A quantitative histochemical assay for activities of mitochondrial electron transport chain complexes in mouse spinal cord sections. J Neurosci Methods 114:165–172PubMedCrossRefGoogle Scholar
  37. Kudin AP, Bimpong-Buta NY, Vielhaber S, Elger CE, Kunz WS (2004) Characterization of superoxide-producing sites in isolated brain mitochondria. J Biol Chem 279:4127–4135PubMedCrossRefGoogle Scholar
  38. Kussmaul L, Hirst J (2006) The mechanism of superoxide production by NADH: ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci USA 103:7607–7612PubMedCrossRefGoogle Scholar
  39. Lee RB, Urban JP (2002) Functional replacement of oxygen by other oxidants in articular cartilage. Arthritis Rheum 46:3190–3200PubMedCrossRefGoogle Scholar
  40. Lehninger AL (1964) The mitochondrion. Molecular basis of structure and function. W.A. Benjamin Inc., New YorkGoogle Scholar
  41. Lenaz G, Fato R, Baracca A, Genova ML (2004) Mitochondrial quinone reductases: complex I. Methods Enzymol 382:3–20PubMedCrossRefGoogle Scholar
  42. Lenaz G, Fato R, Genova ML, Bergamini C, Bianchi C, Biondi A (2006) Mitochondrial complex I: structural and functional aspects. Biochim Biophys Acta 1757:1406–1420PubMedCrossRefGoogle Scholar
  43. Liang HL, Whelan HT, Eells JT, Meng H, Buchmann E, Lerch-Gaggl A, Wong-Riley M (2006) Photobiomodulation partially rescues visual cortical neurons from cyanide-induced apoptosis. Neuroscience 139:639–649PubMedCrossRefGoogle Scholar
  44. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795PubMedCrossRefGoogle Scholar
  45. Miclescu A, Basu S, Wiklund L (2006) Methylene blue added to a hypertonic-hyperoncotic solution increases short-term survival in experimental cardiac arrest. Crit Care Med 34:2806–2813PubMedCrossRefGoogle Scholar
  46. Necula M, Breydo L, Milton S, Kayed R, van der Veer WE, Tone P, Glabe CG (2007) Methylene blue inhibits amyloid Abeta oligomerization by promoting fibrillization. Biochemistry 46:8850–8860PubMedCrossRefGoogle Scholar
  47. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates. Academic Press, San DiegoGoogle Scholar
  48. Perier C, Tieu K, Guegan C, Caspersen C, Jackson-Lewis V, Carelli V, Martinuzzi A, Hirano M, Przedborski S, Vila M (2005) Complex I deficiency primes Bax-dependent neuronal apoptosis through mitochondrial oxidative damage. Proc Natl Acad Sci USA 102:19126–19131PubMedCrossRefGoogle Scholar
  49. Peter C, Hongwan D, Kupfer A, Lauterburg BH (2000) Pharmacokinetics and organ distribution of intravenous and oral methylene blue. Eur J Clin Pharmacol 56:247–250PubMedCrossRefGoogle Scholar
  50. Prusky GT, West PW, Douglas RM (2000) Behavioral assessment of visual acuity in mice and rats. Vision Res 40:2201–2209PubMedCrossRefGoogle Scholar
  51. Riha PD, Bruchey AK, Echevarria DJ, Gonzalez-Lima F (2005) Memory facilitation by methylene blue: dose–dependent effect on behavior and brain oxygen consumption. Eur J Pharmacol 511:151–158PubMedCrossRefGoogle Scholar
  52. Rojas JC, Saavedra JA, Gonzalez-Lima F (2008a) Neuroprotective effects of memantine in a mouse model of retinal degeneration induced by rotenone. Brain Res 1215:208–217PubMedCrossRefGoogle Scholar
  53. Rojas JC, Lee J, John JM, Gonzalez-Lima F (2008b) Neuroprotective effects of near-infrared light in an in vivo model of mitochondrial optic neuropathy. J Neurosci 28:13511–13521PubMedCrossRefGoogle Scholar
  54. Salaris SC, Babbs CF, Voorhees WDIII (1991) Methylene blue as an inhibitor of superoxide generation by xanthine oxidase, A potential new drug for the attenuation of ischemia/reperfusion injury. Biochem Pharmacol 42:499–506PubMedCrossRefGoogle Scholar
  55. Schapira AH, Cooper JM, Dexter D, Clark JB, Jenner P, Marsden CD (1990) Mitochondrial complex I deficiency in Parkinson’s disease. J Neurochem 54:823–827PubMedCrossRefGoogle Scholar
  56. Scott A, Hunter FE Jr (1966) Support of thyroxine-induced swelling of liver mitochondria by generation of high energy intermediates at any one of three sites in electron transport. J Biol Chem 241:1060–1066PubMedGoogle Scholar
  57. Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, Miller GW, Yagi T, Matsuno-Yagi A, Greenamyre JT (2003) Mechanism of toxicity in rotenone models of Parkinson’s disease. J Neurosci 23:10756–10764PubMedGoogle Scholar
  58. Sherer TB, Richardson JR, Testa CM, Seo BB, Panov AV, Yagi T, Matsuno-Yagi A, Miller GW, Greenamyre JT (2007) Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson’s disease. J Neurochem 100:1469–1479PubMedGoogle Scholar
  59. Taniguchi S, Suzuki N, Masuda M, Hisanaga S, Iwatsubo T, Goedert M, Hasegawa M (2005) Inhibition of heparin-induced tau filament formation by phenothiazines, polyphenols, and porphyrins. J Biol Chem 280:7614–7623PubMedCrossRefGoogle Scholar
  60. Tranebjaerg L, Hamel BC, Gabreels FJ, Renier WO, Van Ghelue M (2000) A de novo missense mutation in a critical domain of the X-linked DDP gene causes the typical deafness-dystonia-optic atrophy syndrome. Eur J Hum Genet 8:464–467PubMedCrossRefGoogle Scholar
  61. Valla J, Berndt JD, Gonzalez-Lima F (2001) Energy hypometabolism in posterior cingulate cortex of Alzheimer’s patients: superficial laminar cytochrome oxidase associated with disease duration. J Neurosci 21:4923–4930PubMedGoogle Scholar
  62. Villarreal JS, Gonzalez-Lima F, Berndt J, Barea-Rodriguez EJ (2002) Water maze training in aged rats: effects on brain metabolic capacity and behavior. Brain Res 939:43–51PubMedCrossRefGoogle Scholar
  63. Visarius TM, Stucki JW, Lauterburg BH (1997) Stimulation of respiration by methylene blue in rat liver mitochondria. FEBS Lett 412:157–160PubMedCrossRefGoogle Scholar
  64. Wainwright M, Crossley KB (2002) Methylene blue—a therapeutic dye for all seasons? J Chemother 14:431–443PubMedGoogle Scholar
  65. Wang C, Zhang D, Li G, Liu J, Tian J, Fu F, Liu K (2007) Neuroprotective effects of safflor yellow B on brain ischemic injury. Exp Brain Res 177:533–539PubMedCrossRefGoogle Scholar
  66. Wischik CM, Edwards PC, Lai RY, Roth M, Harrington CR (1996) Selective inhibition of Alzheimer disease-like tau aggregation by phenothiazines. Proc Natl Acad Sci USA 93:11213–11218PubMedCrossRefGoogle Scholar
  67. Wong-Riley M (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res 171:11–28PubMedCrossRefGoogle Scholar
  68. Wong-Riley MT (1989) Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci 12:94–101PubMedCrossRefGoogle Scholar
  69. Wong-Riley MT, Liang HL, Eells JT, Chance B, Henry MM, Buchmann E, Kane M, Whelan HT (2005) Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase. J Biol Chem 280:4761–4771PubMedCrossRefGoogle Scholar
  70. Wright RO, Lewander WJ, Woolf AD (1999) Methemoglobinemia: etiology, pharmacology, and clinical management. Ann Emerg Med 34:646–656PubMedCrossRefGoogle Scholar
  71. Wrubel KM, Riha PD, Maldonado MA, McCollum D, Gonzalez-Lima F (2007) The brain metabolic enhancer methylene blue improves discrimination learning in rats. Pharmacol Biochem Behav 86:712–717PubMedCrossRefGoogle Scholar
  72. Yadava N, Nicholls DG (2007) Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone. J Neurosci 27:7310–7317PubMedCrossRefGoogle Scholar
  73. Zhang X, Jones D, Gonzalez-Lima F (2002) Mouse model of optic neuropathy caused by mitochondrial complex I dysfunction. Neurosci Lett 326:97–100PubMedCrossRefGoogle Scholar
  74. Zhang X, Jones D, Gonzalez-Lima F (2006a) Neurodegeneration produced by rotenone in the mouse retina: a potential model to investigate environmental pesticide contributions to neurodegenerative diseases. J Toxicol Environ Health A 69:1681–1697PubMedCrossRefGoogle Scholar
  75. Zhang X, Rojas JC, Gonzalez-Lima F (2006b) Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res 9:47–57PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Julio C. Rojas
    • 1
  • Joseph M. John
    • 1
  • Jung Lee
    • 1
  • F. Gonzalez-Lima
    • 1
    • 2
  1. 1.Institute for NeuroscienceUniversity of Texas at AustinAustinUSA
  2. 2.Departments of Psychology, Pharmacology and ToxicologyUniversity of Texas at AustinAustinUSA

Personalised recommendations