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.
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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 22:703–712
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 24:308–317
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 3:1301–1306
Borit A (1971) Leigh’s necrotizing encephalomyelopathy, Neuro-ophthalmological abnormalities. Arch Ophthalmol 85:438–442
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 109:33–39
Bruchey AK, Gonzalez-Lima F (2006) Brain activity associated with fear renewal. Eur J Neurosci 24:3567–3577
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 52:183–191
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 332:83–86
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 77:175–181
Carelli V, Ross-Cisneros FN, Sadun AA (2002) Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies. Neurochem Int 40:573–584
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 93:973–983
Chalmers RM, Schapira AH (1999) Clinical, biochemical and molecular genetic features of Leber’s hereditary optic neuropathy. Biochim Biophys Acta 1410:147–158
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):1713–1721
Clark JM, Switzer RL (1977) Experimental biochemistry, 2nd edn. W.H. Freeman and Company, San Francisco
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 67:1852–1854
Degli Esposti M (1998) Inhibitors of NADH-ubiquinone reductase: an overview. Biochim Biophys Acta 1364:222–235
Degli Esposti M, Lenaz G (1982) Kinetics of ubiquinol-1-cytochrome c reductase in bovine heart mitochondria and submitochondrial particles. Biochim Biophys Acta 682:189–200
DiMauro S (1999) Mitochondrial encephalomyopathies: back to Mendelian genetics. Ann Neurol 45:693–694
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–R1267
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 100:3439–3444
Estornell E, Fato R, Pallotti F, Lenaz G (1993) Assay conditions for the mitochondrial NADH: coenzyme Q oxidoreductase. FEBS Lett 332:127–131
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 225–226
Gonzalez-Lima F, Bruchey AK (2004) Extinction memory improvement by the metabolic enhancer methylene blue. Learn Mem 11:633–640
Gonzalez-Lima F, Cada A (1994) Cytochrome oxidase activity in the auditory system of the mouse: a qualitative and quantitative histochemical study. Neuroscience 63:559–578
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 55–90
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 660:34–49
Gundersen HJ, Jensen TB, Osterby R (1978) Distribution of membrane thickness determined by lineal analysis. J Microsc 113:27–43
Harding AJ, Halliday GM, Cullen K (1994) Practical considerations for the use of the optical disector in estimating neuronal number. J Neurosci Methods 51:83–89
Hayes JM, Balkema GW (1993) Elevated dark-adapted thresholds in hypopigmented mice measured with a water maze screening apparatus. Behav Genet 23:395–403
Hevner RF, Wong-Riley MT (1990) Regulation of cytochrome oxidase protein levels by functional activity in the macaque monkey visual system. J Neurosci 10:1331–1340
Hinton DR, Sadun AA, Blanks JC, Miller CA (1986) Optic-nerve degeneration in Alzheimer’s disease. N Engl J Med 315:485–487
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 116:265–280
Hwang JM, Park HW, Kim SJ (1997) Optic neuropathy associated with mitochondrial tRNA[Leu(UUR)] A3243G mutation. Ophthalmic Genet 18:101–105
Ikegami K, Koike T (2003) Non-apoptotic neurite degeneration in apoptotic neuronal death: pivotal role of mitochondrial function in neurites. Neuroscience 122:617–626
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 26:18–24
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 114:165–172
Kudin AP, Bimpong-Buta NY, Vielhaber S, Elger CE, Kunz WS (2004) Characterization of superoxide-producing sites in isolated brain mitochondria. J Biol Chem 279:4127–4135
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 103:7607–7612
Lee RB, Urban JP (2002) Functional replacement of oxygen by other oxidants in articular cartilage. Arthritis Rheum 46:3190–3200
Lehninger AL (1964) The mitochondrion. Molecular basis of structure and function. W.A. Benjamin Inc., New York
Lenaz G, Fato R, Baracca A, Genova ML (2004) Mitochondrial quinone reductases: complex I. Methods Enzymol 382:3–20
Lenaz G, Fato R, Genova ML, Bergamini C, Bianchi C, Biondi A (2006) Mitochondrial complex I: structural and functional aspects. Biochim Biophys Acta 1757:1406–1420
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 139:639–649
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795
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 34:2806–2813
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 46:8850–8860
Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates. Academic Press, San Diego
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 102:19126–19131
Peter C, Hongwan D, Kupfer A, Lauterburg BH (2000) Pharmacokinetics and organ distribution of intravenous and oral methylene blue. Eur J Clin Pharmacol 56:247–250
Prusky GT, West PW, Douglas RM (2000) Behavioral assessment of visual acuity in mice and rats. Vision Res 40:2201–2209
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 511:151–158
Rojas JC, Saavedra JA, Gonzalez-Lima F (2008a) Neuroprotective effects of memantine in a mouse model of retinal degeneration induced by rotenone. Brain Res 1215:208–217
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 28:13511–13521
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 42:499–506
Schapira AH, Cooper JM, Dexter D, Clark JB, Jenner P, Marsden CD (1990) Mitochondrial complex I deficiency in Parkinson’s disease. J Neurochem 54:823–827
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 241:1060–1066
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 23:10756–10764
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 100:1469–1479
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 280:7614–7623
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 8:464–467
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 21:4923–4930
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 939:43–51
Visarius TM, Stucki JW, Lauterburg BH (1997) Stimulation of respiration by methylene blue in rat liver mitochondria. FEBS Lett 412:157–160
Wainwright M, Crossley KB (2002) Methylene blue—a therapeutic dye for all seasons? J Chemother 14:431–443
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 177:533–539
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 93:11213–11218
Wong-Riley M (1979) Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry. Brain Res 171:11–28
Wong-Riley MT (1989) Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci 12:94–101
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 280:4761–4771
Wright RO, Lewander WJ, Woolf AD (1999) Methemoglobinemia: etiology, pharmacology, and clinical management. Ann Emerg Med 34:646–656
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 86:712–717
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 27:7310–7317
Zhang X, Jones D, Gonzalez-Lima F (2002) Mouse model of optic neuropathy caused by mitochondrial complex I dysfunction. Neurosci Lett 326:97–100
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 69:1681–1697
Zhang X, Rojas JC, Gonzalez-Lima F (2006b) Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res 9:47–57
Acknowledgements
This research was supported by CONACYT grant 187413 to Dr. Julio Rojas, and NIH grant MH65728 to Dr. F. Gonzalez-Lima. This study is part of the Ph.D. dissertation of Julio C. Rojas, M.D., and we gratefully acknowledge the comments from Dissertation Committee members: Dr. Timothy Schallert, Dr. Theresa A. Jones, Dr. Andrea Gore, and Dr. Henry G. Rylander.
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Rojas, J.C., John, J.M., Lee, J. et al. Methylene Blue Provides Behavioral and Metabolic Neuroprotection Against Optic Neuropathy. Neurotox Res 15, 260–273 (2009). https://doi.org/10.1007/s12640-009-9027-z
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DOI: https://doi.org/10.1007/s12640-009-9027-z