![Initial lesion (30 mins ADC) and fi nal infarct (24 hrs T2) volumes of vehicle-and MB-treated rats subjected to 60 min middle cerebral artery occlusion. Initial lesion and fi nal infarct volumes of individual animal were connected using dot lines. Mean initial lesion and fi nal infarct were connected using solid lines. Adapted from the reference paper 5]](https://www.researchgate.net/profile/Zhao-Jiang/publication/297896854/figure/fig1/AS:613894871593012@1523375351907/nitial-lesion-30-mins-ADC-and-fi-nal-infarct-24-hrs-T2-volumes-of-vehicle-and_Q320.jpg)
Content uploaded by Zhao Jiang
Author content
All content in this area was uploaded by Zhao Jiang on Nov 08, 2016
Content may be subject to copyright.
Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
Brain
Circulation
Volume 2 | Issue 1 | Jan-Mar 2016
ISSN 2394-8108
Brain Circulation
Publisher: Wolters Kluwer Health - Medknow
Office: B-9, Kanara Business Center, Off Link Rd, Ghatkopar (E), Mumbai-400075, India.
Email: editor@braincirculation.org
Website: http://braincirculation.org
48 © 2016 Brain Circulation | Published by Wolters Kluwer Health – Medknow
Methylene blue treatment
in experimental ischemic stroke:
A mini-review
Zhao Jiang, Timothy Q Duong1
Abstract:
Stroke is among the leading causes of death and long-term disability. Methylene blue (MB), a drug grandfathered
by the Food and Drug Administration with a long history of safe usage in humans for treating methemoglobinemia
and cyanide poisoning, has recently been shown to be neuroprotective in neurodegenerative diseases and brain
injuries. The goal of this paper is to review studies on MB in experimental stroke models.
Key words:
Ischemia, methylene blue (MB), stroke
Introduction
Stroke is the second leading cause of death
and the leading cause of long-term disability
worldwide, and the fourth leading cause of
death in the United States.[1] There are 800,000
new or recurrent strokes per year in the United
States. Of the 6 million Americans who are
stroke survivors, 71% are unable to return to
work. Over $70 billion was expended on stroke
patient care in 2013.[1] This cost is steadily rising
because the conditions that put people at the risk
of stroke (such as heart disease, hypertension,
diabetes, and obesity) are also steadily on the
rise. Recombinant tissue plasminogen activator
(rtPA), the only drug clinically approved to
treat ischemic stroke, is limited to only a small
subset of patients due to the serious risk of often
fatal hemorrhagic transformation and can only
be administered within 4.5 h of stroke onset.[2]
Recently, intraarterial therapy using primarily
stent-retriever technology to achieve mechanical
thrombectomy combined with intravenous (IV)
rtPA administration has been found to be superior
to IV rtPA alone when patients with proximal
cerebral arterial occlusions are treated within 6 h of
symptom onset.[3] Despite the tremendous efforts
taken in stroke research, our ability to minimize
infarct volume and neurological defi cit remains
extremely limited. Thus, there is an urgent need
to develop new treatments for stroke to protect the
brain from the acute phase to the chronic phase.
In acute stroke, a therapeutic approach is
to buy time (i.e., protecting neurons and
glia via sustaining metabolic energy) before
recanalization.[4,5] This may allow the expansion
of the critical treatment time window. During the
reperfusion phase, it is important to minimize
reperfusion injury such as that from excessive
production of reactive oxygen species
[6,7] that
could accelerate mitochondrial damage.[8]
During the chronic phase, the brain undergoes
signifi cant remodeling[9,10] and it is important to
maximize functional recovery. Thus, advanced
drug or reagent methodologies, to enhance
ischemic cells and tissues survival and assist the
effect of thrombolytic treatment, are required in
the development of effective therapies for the
management of stroke patients. Mitochondrial
targeting is one of the promising strategies that
is widely explored.[11,12]
Methylene blue (MB), a Food and Drug
Administration (FDA)-grandfathered
drug, is currently used to treat malaria,
methemoglobinemia, and cyanide poisoning
in humans.[13,14] MB has been rigorously studied
for over 120 years with 5,794 human MB studies
listed in Pubmed (searched in December 2015).
Low-dose MB (1-5 mg/kg IV) is very safe.
Its pharmacokinetics, side effect profile, and
contraindications are well-known and most
importantly minimal in humans.[15,16] There
were only a few negative reports and they were
associated with exceptionally high doses. For
example, MB has been used in parathyroid
Address for
correspondence:
Dr. Zhao Jiang, Research
Imaging Institute,
Radiology, University of
Texas Health Science
Center at San Antonio, San
Antonio, TX, United States.
E-mail: johnjiang406@
gmail.com
Submission: 26-08-2015
Revised: 13-01-2016
Accepted: 20-01-2016
Research Imaging
Institute, Radiology,
1Department of
Ophthalmology,
Radiology and
Physiology, University
of Texas Health
Science Center, San
Antonio, Texas, USA
Access this article online
Quick Response Code:
Website:
http://www.braincirculation.org
DOI:
10.4103/2394-8108.178548
Review Article
How to cite this article: Jiang Z, Duong TQ.
Methylene blue treatment in experimental ischemic
stroke: A mini-review. Brain Circ 2016;2:48-53.
This is an open access arƟ cle distributed under the terms of the
CreaƟ ve Commons AƩ ribuƟ on-NonCommercial-ShareAlike 3.0 License,
which allows others to remix, tweak, and build upon the work
non-commercially, as long as the author is credited and the new
creaƟ ons are licensed under the idenƟ cal terms.
For reprints contact: reprints@medknow.com
Jiang and Duong: A mini-review of methylene blue in ischemia
Brain Circulation - Vol 2, Issue 1, January 2016 49
surgery to aid in lymphatic mapping at doses of 3.5-10 mg/
kg. The FDA also warned physicians about possible serious
serotonin reactions in patients who received IV MB during
parathyroid surgery if taking serotonergic psychiatric drugs.
However, a subsequent report by Mayo Clinic surgeons and
pharmacologists summarized the FDA evidence and literature
and concluded “that the use of methylene blue dye at low
doses for lymphatic mapping likely carries very little risk for
serotonin neurotoxicity.”[17] There has never been any negative
report based on oral MB. Daily 4 mg/kg oral MB has been
used safely for 1 year in clinical trials.[18] MB at 1-3 mg/kg IV
is safely used as a standard treatment for metabolic poisoning
in emergency rooms.
The mechanisms of action of MB are as followed. MB
has renewable auto-oxidizing property, which acts as an
electron cycler that allows MB to redirect electrons to the
mitochondrial electron transport chain, thereby enhancing
adenosine triphosphate (ATP) production and promoting
cell survival. In bypassing complex I-III activity to generate
ATP, MB reduces reactive oxygen species production from
the mitochondrial electron transport chain. The antioxidant
property of MB is thus unique. In vitro studies have fi rmly
established that MB enhances cytochrome c oxidase (complex
IV) activity to produce more ATP in cells under normoxia, and
MB replaces oxygen as the oxidant to sustain ATP generation
under hypoxia while simultaneously reducing oxidative
stress.[19-22] Moreover, chronic MB treatment also modifi es
mitochondrial function and induces long-lasting cellular
changes.[23] Specifi cally, repeated low-dose (0.5-2.0 mg/kg) MB
has long-lasting upregulation of brain cytochrome c oxidase
activity.[20,24-26] MB readily crosses the blood–brain barrier
because of its high lipophilicity.[15]
Low-dose MB has recently been shown to reduce
neurobehavioral impairment in optic neuropathy,[19,27]
traumatic brain injury,[28] Parkinson’s disease,[23,29] Alzheimer
disease,[30-32] and ischemic stroke.[4,5,33] The goal of this article is
to review relevant MB literatures in relation to neuroprotection
in experimental stroke models.
A Pubmed search (December 2015) resulted in 25 papers
relevant to the use of MB in stroke or related to stroke
[Table 1]. Our goal is to review pertinent findings from
most of these.
Basic Stroke-related Methylene Blue Studies
One of the earliest MB experiments was performed by Sidi
et al. in 1987.[34] Arterial pressure transiently increased followed
with MB (5 mg/kg) administration by using hemodynamic
measurements in dogs. Wu and Bohr found the contraction
produced by endothelin was augmented when the intact
aortic rings were treated with MB (10−5 M) in aortas from
Wistar–Kyoto rats but not in those from stroke-prone
spontaneously hypertensive rats.[37] Ishiyama et al. studied the
inhibitory action of MB against nicorandil-induced vasodilation
in dogs.[40] Kontos and Wei demonstrated that MB could
eliminate the arteriolar dilation in response to nitroprusside and
nitroglycerin after permeabilization of the cell membrane.[39]
MB has been shown to increase blood pressure and myocardial
function by inhibiting nitric oxide actions in human septic
shock disease.[41,47,50,52] These studies demonstrated that MB has
vascular effects and causes vasoconstriction transiently, thereby
improving blood pressure, which could help to defend against
hypoperfusion during stroke.
Nitric oxide generation during ischemia and reperfusion plays
a signifi cant role in ischemic and reperfusion injury.[56] There is
evidence that MB decreases or inhibits nitric oxide generation
that might have the potential effect of neuroprotection in
ischemia/reperfusion injury. In order to prove that the
endocardial endothelium of Rana esculenta produces an
amount of nitric oxide that is suffi cient to modulate ventricular
performance, Sys et al. measured the changes of stroke volume
(as a measure of performance in paced frog hearts) and stroke
work (as an index of systolic function) after using MB-induced
inhibition of nitric oxide synthase.[43] This reminded us that MB
could inhibit nitric oxide generation. Evgenov et al. found that
continuous infusion of MB counteracted the early myocardial
dysfunction and derangement of hemodynamics and gas
exchange by the inhibition of nitric oxide pathway in ovine
endotoxemia model.[48]
Xie et al. demonstrated that MB treatment activated 5’adenosine
monophosphate-activated protein kinase signaling but did
not inhibit mammalian target of rapamycin signaling in
serum deprivation cells and normal mouse.[57] This study
suggests that MB-induced neuroprotection is mediated, at
least in part, by macroautophagy. Additionally, MB treatment
altered the levels of microtubule-associated protein light chain
3 type II, cathepsin D, Beclin-1, and p62, suggesting that it was
a potent inducer of autophagy.[58] Thus, MB may be related to
autophagic cell death.
Ryou et al. studied the MB-induced neuroprotective
mechanism focusing on stabilization and activation of
hypoxia-inducible factor-1 in an in vitro oxygen-glucose
deprivation reoxygenation model.[55] They found that MB
activated the erythropoietin-signaling pathway with a
corresponding increase in hypoxia-inducible factor-1 and
consequently related to apoptotic cell death. Together,
these studies shed light on the molecular pathways that MB
modulates.
Methylene Blue Studies in Ischemic Stroke
While low-dose MB has recently been shown to reduce
neurobehavioral impairment in neurodegenerative diseases
(ca. Parkinson’s disease,[23,29] Alzheimer’s disease[30-32]), the
neuroprotective effects of MB on cerebral ischemia in vivo
were only recently demonstrated. In 2006, a Swedish group
found that IV MB at clinical dose was neuroprotective after
experimental cardiac arrest in piglets using histology.[59] Wen
et al. showed that MB could signifi cantly reduce focal cerebral
ischemia reperfusion damage in a transient focal cerebral
ischemia rodent model in 2011 using histology.[60]
Di et al. demonstrated that MB improved neurological
function, and reduced the infarct volume and the necrosis
after acute cerebral ischemic injury by augmenting
mitophagy.[54] These improvements depended on the effect
of MB on mitochondrial structure and function. Acute
cerebral ischemia caused the disorder of and disintegration
Jiang and Duong: A mini-review of methylene blue in ischemia
50 Brain Circulation - Vol 2, Issue 1, January 2016
of mitochondrial structure while MB ameliorated the
destruction of mitochondria. They also further revealed that
the elevation of mitochondrial membrane potential by MB
under oxygen-glucose deprivation conditions mediated the
augmented mitophagy in an oxygen-glucose deprivation
model in vitro.
Shen et al. evaluated the effi cacy of MB to treat ischemic stroke
in a transient middle cerebral artery occlusion model in rats
using noninvasive multimodal magnetic resonance imaging
(MRI).[5] In a randomized double-blinded design in which
vehicle or MB was administered after reperfusion, they found
that the initial lesion volumes defi ned by abnormal apparent
diffusion coeffi cient [61] at 30 min after ischemia were not
signifi cantly different between the two groups. The fi nal infarct
volumes defi ned by T2 changes 2 days after stroke increased
in the vehicle group but decreased in the MB group, yielding
a 30% difference in infarct volume [Figure 1]. Tracking tissue
fate on a pixel-by-pixel basis showed that MB salvaged more
initial ischemic core pixels compared to the control group,
and more mismatch pixels compared to the control group.
This study, for the fi rst time, evaluated the effi cacy of MB
to treat ischemic stroke in rats using longitudinal MRI and
behavioral measures.
Table 1: Published papers about MB related to stroke (searched in Pubmed in December 2015)
Year Cell/animal Dose Function
1987 Dog 1-5 mg/kg Increased arterial pressure transiently[34]
1988 Dog 10-5 M Relaxation of middle cerebral arterial strips was attenuated[35]
1990 Human 10-5 M Inhibited the relaxations induced by thrombin or bradykinin in human
basilar arteries[36]
1990 Rat 10-5 M Augmented the contraction produced by endothelin in intact aortic rings[37]
1991 Feline 10-5 M Inhibited the magnesium defi ciency-related dilations on the tone of middle
cerebral arteries[38]
1993 Cat 5 mM Eliminated the arteriolar dilation after permeabilization of the cell
membrane[39]
1994 Dog 10-5 M Inhibitory action of methylene blue against nicorandil-induced vasodilation
in pial vessels[40]
1995 Human 2 mg/kg Transiently and reproduciblely increased arterial pressure associated with
an improvement in cardiac function[41]
1996 Dog 5 mg/kg Increased arterial pressure, pulmonary arterial pressure, and systemic and
pulmonary vascular resistances but decreased cardiac index and regional
blood fl ow[42]
1997 Frog 10-6 M Inhibition of nitric oxide synthase[43]
1999 Rat 10 MAttenuated endothelium-dependent relaxation in the mesenteric artery[44]
1999 Human 4 mg/kg Increases systemic vascular resistance and may improve myocardial
function[45]
2001 Fish 10-6 M Inhibited nitric oxide synthase[46]
2001 Human 2 mg/kg and 2 mg/kg/h for 1 h Counteracted myocardial depression; maintained oxygen transport and
reduced concurrent adrenergic support[47]
2001 Sheep 10 mg/kg and 2.5 mg/kg/h for 5 h Counteracted the early myocardial dysfunction and derangement of
hemodynamics and gas exchange by inhibiting the nitric oxide pathway[48]
2002 Rat 10-4 and 10-5 M Attenuated endothelium-dependent relaxation in aorta[49]
2002 Human 3 mg/kg Acute vasoconstrictive and positive inotropic effects during septic shock[50]
2005 Human 2 mg/kg Preoperative methylene blue administration reduced the incidence and
severity of vasoplegic syndrome[51]
2010 Human 1 mg/kg, 3 mg/kg, and 7 mg/kg High dose of MB enhanced splanchnic perfusion[52]
2012 HT22 cells 5 MAttenuated superoxide production and antioxidant[53]
2013 Rat 0.5 mg/kg and 1 mg/kg MB treatment minimized ischemic brain injury and improved functional
outcomes.[5]
2014 Rat 1 mg/kg and 3 mg/kg MB delayed the growth rate of the perfusion-diffusion mismatch into
infarction in permanent stroke models[4]
2015 Rat and PC12 cell 1 mg/kg, 5 mg/kg, or 10 mg/kg for
rat and 0.5 M for cell
MB promoted mitophagy by maintaining the MMP§ at a relatively high
level, which contributed to a decrease in necrosis and an improvement
in neurological function, thereby protecting against acute cerebral
ischemic injury[54]
2015 HT22 cells 1 M and 10 MMB protects the hippocampus-derived neuronal cells against OGD†-
reoxygenation injury by enhancing energy metabolism and increasing
HIF-1 protein content accompanied by an activation of the EPO†
signaling pathway.[55]
2015 Rat 1 mg/kg MB induced neuroprotection by enhancing autophagy and reducing
apoptosis in the perfusion-diffusion mismatch tissue following
ischemic stroke[33]
ᴥMB: Methylene blue, §MMP: Matrix metalloproteinase, †OGD: Oxygen-glucose deprivation, ‡EPO: Erythropoietin receptor
Jiang and Duong: A mini-review of methylene blue in ischemia
Brain Circulation - Vol 2, Issue 1, January 2016 51
Rodriguez et al. applied a similar multimodal MRI to test
the hypothesis that MB treatment delays progression of
at-risk tissue (ca. perfusion-diffusion mismatch) to infarct in
permanent middle cerebral artery occlusion in rats at two MB
treatment doses.[4] MB signifi cantly prolonged the perfusion-
diffusion mismatch, and mildly increased the cerebral blood
flow in the hypoperfused tissue. MRI is now a routine
neuroimaging tool in the clinic. MRI plays an important role
in diagnosing, evaluating, and monitoring the cerebral tissue
undergoing stroke and thereby, providing a noninvasive means
to longitudinally evaluate treatment effi cacy.
To further probe the underlying molecular mechanisms of
neuroprotection of MB following transient ischemic stroke in
rats, Jiang et al. employed noninvasive MRI to guide extraction
of the different ischemic tissue types for western blot analysis
of apoptotic and autophaphic cascades.[33] Multimodal MRI
during the acute phase and at 24 h were used to defi ne three
regions of interest (ROIs):
1. The perfusion-diffusion mismatch salvaged by reperfusion,
2. The perfusion-diffusion mismatch not salvaged by
reperfusion, and
3. The ischemic core. The tissues from these ROIs were
extracted for western blot analyses of autophagic and
apoptotic markers.
The major fi ndings were:
1. MB improved cerebral blood fl ow to the perfusion-diffusion
mismatch tissue after reperfusion and minimized harmful
hyperperfusion 24 h after stroke,
2. MB reduces infarct volume and behavioral deficits
following transient ischemic stroke in rats,
3. MB improves cerebral blood fl ow (CBF) to at-risk tissue
after reperfusion and minimizes harmful hyperperfusion
24 h after MCAO,
4. MB inhibits apoptosis and enhances autophagy in the
at-risk tissue but not within the ischemic core,
5. MB modulates the p53-Bax-Bcl2-caspase3 cascade,
inhibiting apoptotic signaling pathways,
6.MB modulates p53-AMPK-TSC2-mTOR cascades,
enhancing autophagic signaling pathways [Figure 2].
Conclusion
Low-dose MB has a long history of safe usage in humans for
treating methemoglobinemia and cyanide poisoning. MB also
has energy-enhancing and antioxidant properties. There are
substantial evidences that MB is neuroprotective for ischemic
stroke. A number of studies have now investigated the
mechanisms of action in ischemic stroke. Noninvasive MRI
offers a means to identify neural correlates of neuroprotection,
target specifi c tissue types for further investigation of molecular
Figure 1: Initial lesion (30 mins ADC) and fi nal infarct (24 hrs T2) volumes of vehicle- and MB-treated rats subjected to 60 min middle cerebral
artery occlusion. Initial lesion and fi nal infarct volumes of individual animal were connected using dot lines. Mean initial lesion and fi nal infarct
were connected using solid lines. Adapted from the reference paper5]
Jiang and Duong: A mini-review of methylene blue in ischemia
52 Brain Circulation - Vol 2, Issue 1, January 2016
mechanisms of action, and longitudinally evaluate treatment
effi cacy. The excellent safety profi le of low-dose MB in humans,
together with noninvasive MRI, could expedite MB stroke
clinical trials. MB treatments could offer novel therapeutic
regimens in combination or alone to improve patient care
following a stroke.
Financial support and sponsorship
This work was supported by NIH/NINDS R01 NS45879.
Confl icts of interest
There are no confl icts of interest.
References
1. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ,
et al.; American Heart Association Statistics Committee and Stroke
Statistics Subcommittee. Heart disease and stroke statistics-2014
update: A report from the American Heart Association.
Circulation 2014;129:e28-292.
2. Davis SM, Donnan GA. 4.5 hours: The new time window for tissue
plasminogen activator in stroke. Stroke 2009;40: 2266-7.
3. Berkhemer OA, Fransen PS, Beumer D, van den Berg LA,
Lingsma HF, Yoo AJ, et al.; MR CLEAN Investigators.
A randomized trial of intraarterial treatment for acute ischemic
stroke. N Engl J Med 2015;372:11-20.
4. Rodriguez P, Jiang Z, Huang S, Shen Q, Duong TQ. Methylene
blue treatment delays progression of perfusion-diffusion
mismatch to infarct in permanent ischemic stroke. Brain Res
2014;1588:144-9.
5. Shen Q, Du F, Huang S, Rodriguez P, Watts LT, Duong TQ.
Neuroprotective effi cacy of methylene blue in ischemic stroke:
An MRI study. PLoS One 2013;8:e79833.
6. Liu Y, Fiskum G, Schubert D. Generation of reactive oxygen
species by the mitochondrial electron transport chain.
J Neurochem 2002;80:780-7.
7. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ.
Production of reactive oxygen species by mitochondria: Central
role of complex III. J Biol Chem 2003;278:36027-31.
8. Crack PJ, Taylor JM. Reactive oxygen species and the modulation
of stroke. Free Radic Biol Med 2005;38:1433-44.
9. Murphy TH, Corbett D. Plasticity during stroke recovery: From
synapse to behaviour. Nat Rev Neurosci 2009;10:861-72.
10. Chopp M, Zhang ZG, Jiang Q. Neurogenesis, angiogenesis,
and MRI indices of functional recovery from stroke. Stroke
2007;38(Suppl):827-31.
11. Chaturvedi RK, Beal MF. Mitochondrial approaches for
neuroprotection. Ann N Y Acad Sci 2008;1147:395-412.
12. Galluzzi L, Blomgren K, Kroemer G. Mitochondrial membrane
permeabilization in neuronal injury. Nat Rev Neurosci
2009;10:481-94.
13. Scheindlin S. Something old... something blue. Mol Interv
2008;8:268-73.
14. Clifton J 2nd, Leikin JB. Methylene blue. Am J Ther 2003;10:289-91.
15. Peter C, Hongwan D, Küpfer A, Lauterburg BH. Pharmacokinetics
and organ distribution of intravenous and oral methylene blue.
Eur J Clin Pharmacol 2000;56:247-50.
16. Walter-Sack I, Rengelshausen J, Oberwittler H, Burhenne J,
Mueller O, Meissner P, et al. High absolute bioavailability of
methylene blue given as an aqueous oral formulation. Eur J Clin
Pharmacol 2009;65:179-89.
17. Shah-Khan MG, Lovely J, Degnim AC. Safety of methylene blue
dye for lymphatic mapping in patients taking selective serotonin
reuptake inhibitors. Am J Surg 2012;204:798-9.
18. Naylor GJ, Martin B, Hopwood SE, Watson Y. A two-year
double-blind crossover trial of the prophylactic effect of methylene
blue in manic-depressive psychosis. Biol Psychiatry 1986;21:915-20.
19. Zhang X, Rojas JC, Gonzalez-Lima F. Methylene blue prevents
neurodegeneration caused by rotenone in the retina. Neurotox
Res 2006;9:47-57.
20. Riha PD, Bruchey AK, Echevarria DJ, Gonzalez-Lima F.
Memory facilitation by methylene blue: Dose-dependent effect
on behavior and brain oxygen consumption. Eur J Pharmacol
2005;511:151-8.
21. Scott A, Hunter FE Jr. 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
1966;241:1060-6.
22. Lindahl PE, Oberg KE. The effect of rotenone on respiration and
its point of attack. Exp Cell Res 1961;23:228-37.
23. Rojas JC, Bruchey AK, Gonzalez-Lima F. Meurometabolic
mechanisms for memory enhancement and neuroprotection of
methylene blue. Prog Neurobiol 2012;96:32-45.
24. Gonzalez-Lima F, Bruchey AK. Extinction memory improvement
by the metabolic enhancer methylene blue. Learn Mem
2004;11:633-40.
25. Wrubel KM, Riha PD, Maldonado MA, McCollum D,
Gonzalez-Lima F. The brain metabolic enhancer methylene blue
improves discrimination learning in rats. Pharmacol Biochem
Behav 2007;86:712-7.
26. Vasquez B, Bieber AL. Direct visualization of IMP — GMP:
Pyrophosphate phosphoribosyltransferase in polyacrylamide
gels. Anal Biochem 1978;84:504-11.
27. Rojas JC, John JM, Lee J, Gonzalez-Lima F. Methylene blue
provides behavioral and metabolic neuroprotection against optic
neuropathy. Neurotox Res 2009;15:260-73.
28. Talley Watts L, Long JA, Chemello J, Van Koughnet S, Fernandez A,
Huang S, et al. Methylene blue is neuroprotective against mild
traumatic brain injury. J Neurotrauma 2014;13: 1063-71.
29. Ishiwata A, Sakayori O, Minoshima S, Mizumura S, Kitamura S,
Katayama Y. Preclinical evidence of Alzheimer changes in
progressive mild cognitive impairment: A qualitative and
quantitative SPECT study. Acta Neurol Scand 2006;114:91-6.
30. Oz M, Lorke DE, Petroianu GA. Methylene blue and Alzheimer’s
disease. Biochem Pharmacol 2009;78:927-32.
31. O’Leary JC 3rd, Li Q, Marinec P, Blair LJ, Congdon EE, Johnson AG,
et al. Phenothiazine-mediated rescue of cognition in tau transgenic
Figure 2: MB induces neuroprotection in neurons following cerebral
ischemia through both the apoptotic p53-Bcl-2-Bax signaling
pathway and autophagic p53-AMPK-TSC2-mTOR signaling
pathway. The arrows show the effect of MB on these pathways.
Adapted from the reference paper[33]
Jiang and Duong: A mini-review of methylene blue in ischemia
Brain Circulation - Vol 2, Issue 1, January 2016 53
mice requires neuroprotection and reduced soluble tau burden.
Mol Neurodegener 2010;5:45.
32. Medina DX, Caccamo A, Oddo S. Methylene blue reduces a
levels and rescues early cognitive defi cit by increasing proteasome
activity. Brain Pathol 2011;21:140-9.
33. Jiang Z, Watts LT, Huang S, Shen Q, Rodriguez P, Chen C, et al.
The effects of methylene blue on autophagy and apoptosis in
mri-defi ned normal tissue, ischemic penumbra and ischemic core.
PLoS One 2015;10:e0131929.
34. Sidi A, Paulus DA, Rush W, Gravenstein N, Davis RF. Methylene
blue and indocyanine green artifactually lower pulse oximetry
readings of oxygen saturation. Studies in dogs. J Clin Monit
1987;3:249-56.
35. Onoue H, Nakamura N, Toda N. Endothelium-dependent and
-independent responses to vasodilators of isolated dog cerebral
arteries. Stroke 1988;19:1388-94.
36. Hatake K, Kakishita E, Wakabayashi I, Sakiyama N, Hishida S.
Effect of aging on endothelium-dependent vascular relaxation of
isolated human basilar artery to thrombin and bradykinin. Stroke
1990;21:1039-43.
37. Wu CC, Bohr DF. Role of endothelium in the response to
endothelin in hypertension. Hypertension 1990;16:677-81.
38. Szabó C, Faragó M, Dóra E, Horváth I, Kovách AG. Endothelium-
dependent infl uence of small changes in extracellular magnesium
concentration on the tone of feline middle cerebral arteries. Stroke
1991;22:785-9.
39. Kontos HA, Wei EP. Hydroxyl radical-dependent inactivation of
guanylate cyclase in cerebral arterioles by methylene blue and by
LY83583. Stroke 1993;24:427-34.
40. Ishiyama T, Dohi S, Iida H, Akamatsu S, Ohta S, Shimonaka H.
Mechanisms of vasodilation of cerebral vessels induced by
the potassium channel opener nicorandil in canine in vivo
experiments. Stroke 1994;25:1644-50.
41. Preiser JC, Lejeune P, Roman A, Carlier E, De Backer D,
Leeman M, et al. Methylene blue administration in septic shock:
A clinical trial. Crit Care Med 1995;23:259-64.
42. Zhang H, Rogiers P, Friedman G, Preiser JC, Spapen H,
Buurman WA, et al. Effects of nitric oxide donor SIN-1 on oxygen
availability and regional blood fl ow during endotoxic shock.
Arch Surg 1996;131:767-74.
43. Sys SU, Pellegrino D, Mazza R, Gattuso A, Andries LJ, Tota L.
Endocardial endothelium in the avascular heart of the frog:
Morphology and role of nitric oxide. J Exp Biol 1997;200:3109-18.
44. Sunano S, Watanabe H, Tanaka S, Sekiguchi F, Shimamura K.
Endothelium-derived relaxing, contracting and hyperpolarizing
factors of mesenteric arteries of hypertensive and normotensive
rats. Br J Pharmacol 1999;126:709-16.
45. Weingartner R, Oliveira E, Oliveira ES, Sant’Anna UL,
Oliveira RP, Azambuja LA, et al. Blockade of the action of
nitric oxide in human septic shock increases systemic vascular
resistance and has detrimental effects on pulmonary function
after a short infusion of methylene blue. Braz J Med Biol Res
1999;32:1505-13.
46. Imbrogno S, De Iuri L, Mazza R, Tota B. Nitric oxide modulates
cardiac performance in the heart of Anguilla anguilla. J Exp Biol
2001;204:1719-27.
47. Kirov MY, Evgenov OV, Evgenov NV, Egorina EM,
Sovershaev MA, Sveinbjørnsson B, et al. Infusion of methylene
blue in human septic shock: A pilot, randomized, controlled
study. Crit Care Med 2001;29:1860-7.
48. Evgenov OV, Sveinbjørnsson B, Bjertnaes LJ. Continuously
infused methylene blue modulates the early cardiopulmonary
response to endotoxin in awake sheep. Acta Anaesthesiol Scand
2001;45:1246-54.
49. Sekiguchi F, Miyake Y, Kashimoto T, Sunano S. Unaltered
caffeine-induced relaxation in the aorta of stroke-prone
spontaneously hypertensive rats (SHRSP). J Smooth Muscle Res
2002 38:11-22.
50. Donati A, Conti G, Loggi S, Münch C, Coltrinari R, Pelaia P, et al.
Does methylene blue administration to septic shock patients
affect vascular permeability and blood volume? Crit Care Med
2002;30:2271-7.
51. Ozal E, Kuralay E, Yildirim V, Kilic S, Bolcal C, Kücükarslan N,
et al. Preoperative methylene blue administration in patients at
high risk for vasoplegic syndrome during cardiac surgery. Ann
Thorac Surg 2005;79:1615-9.
52. Juffermans NP, Vervloet MG, Daemen-Gubbels CR, Binnekade JM,
de Jong M, Groeneveld AB. A dose-fi nding study of methylene
blue to inhibit nitric oxide actions in the hemodynamics of human
septic shock. Nitric Oxide 2010;22:275-80.
53. Poteet E, Winters A, Yan LJ, Shufelt K, Green KN, Simpkins JW,
et al. Neuroprotective actions of methylene blue and its
derivatives. PLoS One 2012;7:e48279.
54. Di Y, He YL, Zhao T, Huang X, Wu KW, Liu SH, et al. Methylene
blue reduces acute cerebral ischemic injury via the induction of
mitophagy. Mol Med 2015;21:420-9.
55. Ryou MG, Choudhury GR, Li W, Winters A, Yuan F, Liu R, et al.
Methylene blue-induced neuronal protective mechanism against
hypoxia-reoxygenation stress. Neuroscience 2015;301:193-203.
56. Gürsoy-Ozdemir Y, Bolay H, Saribaş O, Dalkara T. Role of
endothelial nitric oxide generation and peroxynitrite formation
in reperfusion injury after focal cerebral ischemia. Stroke
2000;31:1974-81.
57. Xie L, Li W, Winters A, Yuan F, Jin K, Yang S. Methylene blue
induces macroautophagy through 5’ adenosine monophosphate-
activated protein kinase pathway to protect neurons from serum
deprivation. Front Cell Neurosci 2013;7:56.
58. Congdon EE, Wu JW, Myeku N, Figueroa YH, Herman M,
Marinec PS, et al. Methylthioninium chloride (methylene blue)
induces autophagy and attenuates tauopathy in vitro and in vivo.
Autophagy 2012;8:609-22.
59. Sharma HS, Miclescu A, Wiklund L. Cardiac arrest-induced
regional blood-brain barrier breakdown, edema formation and
brain pathology: A light and electron microscopic study on a new
model for neurodegeneration and neuroprotection in porcine
brain. J Neural Transm (Vienna) 2011;118:87-114.
60. Wen Y, Li W, Poteet EC, Xie L, Tan C, Yan LJ, et al. Alternative
mitochondrial electron transfer as a novel strategy for
neuroprotection. J Biol Chem 2011;286:16504-15.
61. Moseley ME, Cohen Y, Mintorovitch J, Chileuitt L, Shimizu H,
Kucharczyk J, et al. Early detection of regional cerebral ischemia
in cats: Comparison of diffusion- and T2-weighted MRI and
spectroscopy. Magn Reson Med 1990;14:330-46.
