Methylene blue as a cerebral metabolic and hemodynamic enhancer

Ai-Ling Lin, Ethan Poteet, Fang Du, Roy C Gourav, Ran Liu, Yi Wen, Andrew Bresnen, Shiliang Huang, Peter T Fox, Shao-Hua Yang, Timothy Q Duong, Ai-Ling Lin, Ethan Poteet, Fang Du, Roy C Gourav, Ran Liu, Yi Wen, Andrew Bresnen, Shiliang Huang, Peter T Fox, Shao-Hua Yang, Timothy Q Duong

Abstract

By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson's and Alzheimer's diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB's effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO(2)) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I-III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO(2) under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer's and Parkinson's diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive, nondestructive way to evaluate treatment efficacy.

Conflict of interest statement

Competing Interests: The University of North Texas Health Science Center has filed a PCT patent application entitled “Compounds that enable alternative mitochondrial electron transfer”. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. MB enhances mitochondrial complex I–III…
Figure 1. MB enhances mitochondrial complex I–III activity.
MB enhances mitochondrial complex I–III activity (A), but not II–III (B) activity in mitochondria isolated from rat brains. Antimycin A, an inhibitor of complex III, significantly reduced complex I–III and II–III activities. Data are mean ± SD; ***, p<0.001.
Figure 2. MB increases OCR in HT-22…
Figure 2. MB increases OCR in HT-22 cells.
(A) We monitored cellular OCR with sequential administration of MB/vehicle, oligomycin, FCCP, and rotenone. Quantitative analysis of OCR shows that MB increases OCR under (B) normal, (C) ATP synthase inhibition (oligomycin), (D) FCCP, and (E) complex I inhibition (rotenone). Data are mean ± SD; ***, p<0.001.
Figure 3. MB increases glucose uptake in…
Figure 3. MB increases glucose uptake in HT-22 cells.
Representative live cell images show glucose uptake in (A) vehicle- and (B) MB-treated HT-22 cells. (C) Quantitative analysis indicates a significant increase of glucose uptake upon MB treatment at 10 µM. Data are mean ± SD; ***, p<0.001.
Figure 4. MB enhances glucose uptake and…
Figure 4. MB enhances glucose uptake and CMRO2 under normoxic conditions.
(A) Averaged values of glucose uptake (GU). (B) Glucose uptake maps from a single rat under control (normoxia) and MB (normoxia + MB) conditions. (C) Averaged values of global CMRO2. (D) Averaged values of OEF. (E) Arterial (SO2,a) and venous (SO2,v) oxygenation. (F) Oxygen content (CaO2). Data are mean ± SD; ***, p<0.001.
Figure 5. MB enhances global and regional…
Figure 5. MB enhances global and regional CBF under normoxic
conditions. (A) Brain regions related to PD and AD pathology and the CBF maps under control (normoxia) and MB (normoxia + MB) conditions. Red, cingulate cortex; green, motor cortex; yellow, frontoparietal cortex; blue, hippocampus. (B) Global CBF and regional CBF in cingulate, motor, frontoparietal cortices and hippocampus. Data are mean ± SD; **, p<0.01.
Figure 6. Glucose uptake, CBF, and CMRO…
Figure 6. Glucose uptake, CBF, and CMRO2 were reduced under hypoxic conditions but enhanced by MB treatment.
Comparison between normoxia and hypoxia of (A) glucose uptake, (B) CBF, (C) OEF, and (D) CMRO2. Comparison between control (hypoxia) and MB treatment (hypoxia + MB) of (E, F) glucose uptake (values and maps). (G, H) CBF (values and maps), (I) OEF, (J) CMRO2, (K) arterial (SO2,a) and venous (SO2,v) oxygenation. (L) Oxygen content (CaO2). Data are mean ± SD; ***, p<0.001.
Figure 7. MB did not affect PO…
Figure 7. MB did not affect PO2, PCO2, and hematocrit (Hct).
(A) PO2 decreased under hypoxia but did not change with MB treatment (under either condition). (B) PCO2 increased under hypoxia but did not change with MB treatment (under either condition). (C) Hematocrit was not affected by gas type or MB treatment. Data are mean ± SD; ***, p<0.001; ns, not significant.

References

    1. Fukui H, Moraes CT (2008) The mitochondrial impairment, oxidative stress and neurodegeneration connection: reality or just an attractive hypothesis? Trends Neurosci 31: 251–256.
    1. Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS, et al. (1977) The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 28: 897–916.
    1. Cunnane S, Nugent S, Roy M, Courchesne-Loyer A, Croteau E, et al. (2011) Brain fuel metabolism, aging, and Alzheimer’s disease. Nutrition 27: 3–20.
    1. Hoyer S (1991) Abnormalities of glucose metabolism in Alzheimer’s disease. Ann N Y Acad Sci 640: 53–58.
    1. Nagata K, Buchan RJ, Yokoyama E, Kondoh Y, Sato M, et al. (1997) Misery perfusion with preserved vascular reactivity in Alzheimer’s disease. Ann N Y Acad Sci 826: 272–281.
    1. Scheindlin S (2008) Something old… something blue. Mol Interv 8: 268–273.
    1. Wainwright M, Crossley KB (2002) Methylene Blue–a therapeutic dye for all seasons? J Chemother 14: 431–443.
    1. Atamna H, Nguyen A, Schultz C, Boyle K, Newberry J, et al. (2008) Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J 22: 703–712.
    1. Tokumitsu Y, Ui M (1973) The incorporation of 32 P i into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation. Biochim Biophys Acta 292: 310–324.
    1. Wen Y, Li W, Poteet EC, Xie L, Tan C, et al. (2011) Alternative mitochondrial electron transfer as a novel strategy for neuroprotection. J Biol Chem 286: 16504–16515.
    1. Lindahl PE, Oberg KE (1961) The effect of rotenone on respiration and its point of attack. Exp Cell Res 23: 228–237.
    1. 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.
    1. Zhang X, Rojas JC, Gonzalez-Lima F (2006) Methylene blue prevents neurodegeneration caused by rotenone in the retina. Neurotox Res 9: 47–57.
    1. Oz M, Lorke DE, Petroianu GA (2009) Methylene blue and Alzheimer’s disease. Biochem Pharmacol 78: 927–932.
    1. O’Leary JC 3rd, Li Q, Marinec P, Blair LJ, Congdon EE, et al (2010) Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden. Mol Neurodegener 5: 45.
    1. Medina DX, Caccamo A, Oddo S (2011) Methylene blue reduces abeta levels and rescues early cognitive deficit by increasing proteasome activity. Brain Pathol 21: 140–149.
    1. Ishiwata A, Sakayori O, Minoshima S, Mizumura S, Kitamura S, et al. (2006) Preclinical evidence of Alzheimer changes in progressive mild cognitive impairment: a qualitative and quantitative SPECT study. Acta Neurol Scand 114: 91–96.
    1. Minoshima S, Giordani B, Berent S, Frey KA, Foster NL, et al. (1997) Metabolic reduction in the posterior cingulate cortex in very early Alzheimer’s disease. Ann Neurol 42: 85–94.
    1. Riha PD, Rojas JC, Gonzalez-Lima F (2011) Beneficial network effects of methylene blue in an amnestic model. Neuroimage 54: 2623–2634.
    1. 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.
    1. Rojas JC, Bruchey AK, Gonzalez-Lima F (2012) Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue. Prog Neurobiol 96: 32–45.
    1. Natarajan A, Srienc F (1999) Dynamics of glucose uptake by single Escherichia coli cells. Metab Eng 1: 320–333.
    1. Louters LL, Dyste SG, Frieswyk D, Tenharmsel A, Vander Kooy TO, et al. (2006) Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells. Life Sci 78: 586–591.
    1. Powers WJ, Videen TO, Markham J, Walter V, Perlmutter JS (2011) Metabolic control of resting hemispheric cerebral blood flow is oxidative, not glycolytic. J Cereb Blood Flow and Metab 31: 1223–1228.
    1. Raichle ME, MacLeod AM, Snyder AZ, Powers WJ, Gusnard DA, et al. (2001) A default mode of brain function. Proc Natl Acad Sci U S A 98: 676–682.
    1. Sicard KM, Duong TQ (2005) Effects of hypoxia, hyperoxia, and hypercapnia on baseline and stimulus-evoked BOLD, CBF, and CMRO2 in spontaneously breathing animals. Neuroimage 25: 850–858.
    1. Abate MG, Trivedi M, Fryer TD, Smielewski P, Chatfield DA, et al. (2008) Early derangements in oxygen and glucose metabolism following head injury: the ischemic penumbra and pathophysiological heterogeneity. Neurocrit Care 9: 319–325.
    1. Zhu J, Chu CT (2010) Mitochondrial dysfunction in Parkinson’s disease. J Alzheimers Dis 20 Suppl 2 S325–334.
    1. Butterfield DA, Perluigi M, Sultana R (2006) Oxidative stress in Alzheimer’s disease brain: new insights from redox proteomics. Eur J Pharmacol 545: 39–50.
    1. Bruchey AK, Gonzalez-Lima F (2008) Behavioral, Physiological and Biochemical Hormetic Responses to the Autoxidizable Dye Methylene Blue. Am J Pharmacol Toxicol 3: 72–79.
    1. Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55: 698–707.
    1. Dickinson DB, Misch MJ, Drury RE (1970) Freezing damage to isolated tomato fruit mitochondria as modified by cryoprotective agents and storage temperature. Plant Physiol 46: 200–203.
    1. Lee WC, Chang CH, Ho CL, Chen LC, Wu YH, et al. (2011) Early detection of tumor response by FLT/microPET Imaging in a C26 murine colon carcinoma solid tumor animal model. J Biomed Biotechnol 2011: 535902.
    1. Duong TQ, Silva AC, Lee SP, Kim SG (2000) Functional MRI of calcium-dependent synaptic activity: cross correlation with CBF and BOLD measurements. Magn Reson Med 43: 383–392.
    1. Shen Q, Ren H, Cheng H, Fisher M, Duong TQ (2005) Functional, perfusion and diffusion MRI of acute focal ischemic brain injury. J Cereb Blood Flow and Metab 25: 1265–1279.
    1. 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.

Source: PubMed

Patrocinadores y Colaboradores

Condiciones médicas

Intervenciones de drogas

3
Suscribir