ArticlePDF Available

Methylene Blue Preserves Cytochrome Oxidase Activity and Prevents Neurodegeneration and Memory Impairment in Rats With Chronic Cerebral Hypoperfusion

Authors:

Abstract and Figures

Chronic cerebral hypoperfusion in neurocognitive disorders diminishes cytochrome oxidase activity leading to neurodegenerative effects and impairment of learning and memory. Methylene blue at low doses stimulates cytochrome oxidase activity and may thus counteract the adverse effects of cerebral hypoperfusion. However, the effects of methylene blue on cytochrome oxidase activity during chronic cerebral hypoperfusion have not been described before. To test this hypothesis, rats underwent bilateral carotid artery occlusion or sham surgery, received daily 4 mg/kg methylene blue or saline injections, and learned a visual water task. Brain mapping of cytochrome oxidase activity was done by quantitative enzyme histochemistry. Permanent carotid occlusion for 1 month resulted in decreased cytochrome oxidase activity in visual cortex, prefrontal cortex, perirhinal cortex, hippocampus and amygdala, and weaker interregional correlation of cytochrome oxidase activity between these regions. Methylene blue preserved cytochrome oxidase activity in regions affected by carotid occlusion and strengthened their interregional correlations of cytochrome oxidase activity, which prevented neurodegenerative effects and facilitated task-specific learning and memory. Brain-behavior correlations revealed positive correlations between performance and brain regions in which cytochrome oxidase activity was preserved by methylene blue. These results are the first to demonstrate that methylene blue prevents neurodegeneration and memory impairment by preserving cytochrome oxidase activity and interregional correlation of cytochrome oxidase activity in brain regions susceptible to chronic hypoperfusion. This demonstration provides further support for the hypothesis that lower cerebral blood flow results in an Alzheimer’s-like syndrome and that stimulating cytochrome oxidase activity with low-dose methylene blue is neuroprotective.
Content may be subject to copyright.
fncel-14-00130 May 18, 2020 Time: 14:3 # 1
ORIGINAL RESEARCH
published: 20 May 2020
doi: 10.3389/fncel.2020.00130
Edited by:
Karla Guadalupe Carvajal,
National Institute of Pediatrics
(Mexico), Mexico
Reviewed by:
Jon Storm-Mathisen,
University of Oslo, Norway
Bryan Victor Phillips-Farfan,
National Institute of Pediatrics
(Mexico), Mexico
*Correspondence:
F. Gonzalez-Lima
gonzalezlima@utexas.edu
Specialty section:
This article was submitted to
Cellular Neuropathology,
a section of the journal
Frontiers in Cellular Neuroscience
Received: 25 April 2019
Accepted: 20 April 2020
Published: 20 May 2020
Citation:
Auchter AM, Barrett DW,
Monfils MH and Gonzalez-Lima F
(2020) Methylene Blue Preserves
Cytochrome Oxidase Activity
and Prevents Neurodegeneration
and Memory Impairment in Rats With
Chronic Cerebral Hypoperfusion.
Front. Cell. Neurosci. 14:130.
doi: 10.3389/fncel.2020.00130
Methylene Blue Preserves
Cytochrome Oxidase Activity and
Prevents Neurodegeneration and
Memory Impairment in Rats With
Chronic Cerebral Hypoperfusion
Allison M. Auchter, Douglas W. Barrett, Marie H. Monfils and F. Gonzalez-Lima*
Department of Psychology, Institute for Neuroscience, The University of Texas at Austin, Austin, TX, United States
Chronic cerebral hypoperfusion in neurocognitive disorders diminishes cytochrome
oxidase activity leading to neurodegenerative effects and impairment of learning and
memory. Methylene blue at low doses stimulates cytochrome oxidase activity and
may thus counteract the adverse effects of cerebral hypoperfusion. However, the
effects of methylene blue on cytochrome oxidase activity during chronic cerebral
hypoperfusion have not been described before. To test this hypothesis, rats
underwent bilateral carotid artery occlusion or sham surgery, received daily 4 mg/kg
methylene blue or saline injections, and learned a visual water task. Brain mapping
of cytochrome oxidase activity was done by quantitative enzyme histochemistry.
Permanent carotid occlusion for 1 month resulted in decreased cytochrome oxidase
activity in visual cortex, prefrontal cortex, perirhinal cortex, hippocampus and amygdala,
and weaker interregional correlation of cytochrome oxidase activity between these
regions. Methylene blue preserved cytochrome oxidase activity in regions affected
by carotid occlusion and strengthened their interregional correlations of cytochrome
oxidase activity, which prevented neurodegenerative effects and facilitated task-specific
learning and memory. Brain-behavior correlations revealed positive correlations between
performance and brain regions in which cytochrome oxidase activity was preserved
by methylene blue. These results are the first to demonstrate that methylene blue
prevents neurodegeneration and memory impairment by preserving cytochrome oxidase
activity and interregional correlation of cytochrome oxidase activity in brain regions
susceptible to chronic hypoperfusion. This demonstration provides further support for
the hypothesis that lower cerebral blood flow results in an Alzheimer’s-like syndrome
and that stimulating cytochrome oxidase activity with low-dose methylene blue
is neuroprotective.
Keywords: methylene blue, chronic cerebral hypoperfusion, carotid artery occlusion, cytochrome oxidase,
interregional correlations, vascular hypothesis of Alzheimer’s dementia
Frontiers in Cellular Neuroscience | www.frontiersin.org 1May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 2
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
INTRODUCTION
Aging and dementia involve progressive reduction in cerebral
blood flow and energy metabolism that result in cognitive
dysfunction (de la Torre, 1999;Farkas and Luiten, 2001). Patients
with cerebrovascular disorders and late-onset Alzheimer’s disease
(AD) show marked cerebral hypoperfusion (de la Torre, 2012).
Even normal aging-related reduction in cerebral blood flow
results in significant functional pathology when combined with
other factors such as cardiovascular disease (Haley et al.,
2007) and cerebrovascular ischemia (de la Torre et al., 1997;
Cada et al., 2000). Interestingly, in AD patients who have
not suffered cerebral infarction, cerebral perfusion is reduced
more symmetrically, globally and chronically than in stroke
patients (Tachibana et al., 1984), implicating chronic cerebral
hypoperfusion in the pathogenesis of AD (de la Torre, 2018).
Permanent bilateral carotid artery occlusion (2-vessel
occlusion; 2VO) is a useful model for the reproduction of
chronic cerebral hypoperfusion as it occurs in human aging
and AD (de la Torre et al., 1992;de la Torre, 1999, 2000;Farkas
et al., 2007). Typically, in rats permanent vessel occlusion does
not result in reperfusion injury (as a result of instant recovery
of perfusion). Additionally, cerebral hypoperfusion is global,
damage to nervous tissue is less dramatic, and there are no
obvious signs of motor dysfunction or seizures (de la Torre et al.,
1992;Farkas et al., 2007). When neurons are starved of glucose
and oxygen via chronic cerebral hypoperfusion, the impending
result is mitochondrial dysfunction. If mitochondria do not
receive enough glucose and oxygen, electrons from the electron
transport chain used to drive ATP synthesis are taken up by
other molecules, resulting in the formation of damaging reactive
oxygen species (Rojas et al., 2012).
We hypothesized that certain properties of low-dose
methylene blue (MB) could be neuroprotective under conditions
of reduced supply of glucose and oxygen such as in chronic
cerebral hypoperfusion (Gonzalez-Lima and Auchter, 2015).
Under physiological conditions in nervous tissue, nearly all the
electrons donated to the electron transport chain are derived
from the transformation of glucose for the generation of the
electron donors NADH and FADH (Erecinska and Silver, 1989).
However, MB at low doses reaches a redox equilibrium inside
mitochondria that allows MB to cycle electrons directly into
the electron transport chain (Rojas et al., 2012). In this way,
MB could compensate for a reduced supply of glucose to the
brain by becoming an alternative source of electrons donated
to the electron transport chain. Cytochrome oxidase (CO) is
the last enzyme in the electron transport chain that passes the
electrons to oxygen as the final electron acceptor (Erecinska and
Silver, 1989). However, there is evidence that MB can maintain
energy production even under hypoxic conditions (Lee and
Urban, 2002) because MB can replace oxygen by accepting
electrons from CO at the end of the electron transport chain
(Rojas et al., 2012). Therefore, maintaining CO activity by
MB’s electron cycling could compensate for 2VO reducing both
glucose and oxygen.
Neuropathological changes that result from 2VO resemble
those of late-onset AD, strengthening its use as an experimental
model (de la Torre, 1999, 2000). Since hippocampal damage is a
feature of AD, and the hippocampus is also particularly sensitive
to ischemia, the time course of damage to the hippocampus
as a result of 2VO surgery has been well characterized. While
conspicuous hippocampal damage is not seen during the first
week after 2VO (Ohtaki et al., 2006), damage to the CA1 subfield
is observed in 629% of animals at 2 weeks (Schmidt-Kastner
et al., 2001;Farkas et al., 2006), 55% at 4 weeks (Ohtaki et al.,
2006), and total hippocampal destruction was observed in 67% of
2VO rats at 813 weeks (Farkas et al., 2004;Liu et al., 2006).
2VO also serves as a good model for cognitive aging and
dementia because it not only results in global neurological
changes, but also results in learning and memory impairment
(Cada et al., 2000;de la Torre, 2000). It has been well established
that experimental cerebral hypoperfusion compromises spatial
learning in rats (Farkas and Luiten, 2001;Liu et al., 2005;Shang
et al., 2005). Non-spatial object recognition deficits have also been
observed 60 and 90 days following 2VO surgery (Sarti et al.,
2002), but we did not observe deficits in odor recognition after
30 days (Auchter et al., 2014).
Methylene blue is a blue dye with neurometabolic enhancing
properties at low doses (Rojas et al., 2012). MB crosses the
blood-brain barrier and diffuses into the mitochondrial matrix,
where at low doses it forms a redox equilibrium with the
enzymes of the electron transport chain (Bruchey and Gonzalez-
Lima, 2008;Riha et al., 2011). MB enhances mitochondrial
respiration, chiefly by increasing the activity of cytochrome
oxidase (CO) (Rojas et al., 2012). CO is the terminal enzyme
of the electron transport chain and its activity reflects neuronal
activity (Wong-Riley, 1989), including neuronal activity of
circuits involved in rat water maze tasks (Villarreal et al., 2002;
Conejo et al., 2010). MB enhancement of CO activity is coupled
with increases in ATP production and oxygen consumption
(Riha et al., 2005;Atamna et al., 2008;Bruchey and Gonzalez-
Lima, 2008). Through enhanced mitochondrial efficiency, MB
also reduces the incidence of reactive oxygen species formation
(Salaris et al., 1991) and consequently delays cellular senescence
(Atamna et al., 2008).
Methylene blue at low doses has been shown to enhance both
spatial (Callaway et al., 2002;Riha et al., 2005;Wrubel et al., 2007)
and non-spatial (Gonzalez-Lima and Bruchey, 2004) memories,
and such enhancement is also marked by increases in CO activity
(Callaway et al., 2002, 2004). In humans, MB enhances various
memory tasks and modulates brain functional connectivity
(Telch et al., 2014;Rodriguez et al., 2016, 2017;Zoellner et al.,
2017). MB has also been shown to be neuroprotective in rodent
models of stroke (Salaris et al., 1991;Miclescu et al., 2010;
Shen et al., 2013), hypoxia (Huang et al., 2013), Alzheimer’s
disease (Callaway et al., 2002;Riha et al., 2005), Parkinson’s
disease (Rojas et al., 2009b;Smith et al., 2017), and mitochondrial
optic neuropathy (Zhang et al., 2006;Rojas et al., 2009a).
However, MB effects have not been described during chronic
cerebral hypoperfusion.
The objectives of this experiment were to describe (1) how
2VO weakens brain regional cytochrome oxidase activity as
measured by quantitative enzyme histochemistry, interregional
correlation of cytochrome oxidase activity and performance
Frontiers in Cellular Neuroscience | www.frontiersin.org 2May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 3
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
on a visual discrimination task, (2) how MB strengthens
performance and preserves both regional cytochrome oxidase
activity and interregional correlation of cytochrome oxidase
activity, and (3) how specific changes in brain regional
cytochrome oxidase activity are correlated with behavioral
performance (Auchter, 2015).
Our hypothesis was that chronic cerebral hypoperfusion
results in reduction in regional CO activity in brain regions
susceptible to hypoxia and a reduction in interregional
correlation of cytochrome oxidase activity between regions
involved in visual discrimination learning. Treatment of chronic
cerebral hypoperfusion with daily low-dose methylene blue
(MB) injections may restore both brain regional activity
and interregional correlation of cytochrome oxidase activity,
preventing neurodegeneration and memory impairment.
MATERIALS AND METHODS
Subjects
Subjects for this brain analysis were the same subjects used in the
behavioral study of Auchter et al. (2014). Subjects were 39 adult
male Long-Evans rats weighing approximately 500600 g at the
time of surgery. Two rats died from surgical complications and
one rat died of a respiratory infection during visual water task
training. Additionally, it was determined upon brain extraction
that one subject suffered a hemorrhagic stroke, and thus was
excluded from the analysis, making the final N= 35.
Ethical and Biosafety Measures
Rats were raised from birth in Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC)-approved
facilities under standard laboratory conditions (12 h: 12 h,
light: dark cycle) with ad libitum access to food and water.
All animal care and experimental procedures were approved by
the University of Texas at Austin’s Institutional Animal Care
and Use Committee (IACUC), and followed NIH guidelines as
described in the Guide for the Care and Use of Laboratory
Animals, 8th edition. All animal research was conducted in the
Animal Resources Center at the University of Texas at Austin,
an AAALAC-accredited facility which is inspected twice a year
by UT’s IACUC to ensure compliance with all relevant laws,
regulations, and policies regarding animal care and use. Personal
protective equipment (PPE) such as lab coats, gloves, and face
masks were used for all animal interactions. Cytochrome oxidase
histochemistry was performed in the histochemical suite of the
PI’s lab in the Animal Resources Center, which is inspected
and approved twice a year by UT’s Environmental Health
and Safety department. PPE was used during all stages of the
histochemical staining procedure, and all chemical storage and
usage complied with NIH guidelines as described in the NIH
Chemical Safety Guide (2015).
Surgical Procedures and Interventions
Auchter et al. (2014) provides detailed explanations of the
surgical and behavioral procedures, and the experimental design
is shown in Figure 1. Briefly, the experiment followed a 2 ×2
FIGURE 1 | Experimental design and timeline. After 2VO or sham surgery
(Day 0), rats received daily intraperitoneal injections of 4 mg/kg MB or saline
(2 ×2 design = four groups). Rats recovered for 1 week (Days 1–7) before the
start of behavioral training and testing (Days 8–30). VWT, visual water maze
task; ED, elemental pattern discrimination; TP, transverse pattern
discrimination; sac, sacrifice on Day 31 to collect brains for analysis.
experimental design, whereby subjects were randomly assigned
to groups which were given either 2VO surgery or a sham control
(without vessel occlusion), followed by daily intraperitoneal
injections of either 4 mg/kg MB or an equivalent volume of saline.
Before surgery, we anesthetized the rats beginning with
4% isoflurane inhalation, followed by general anesthesia using
1.53% during the surgery, with an E-Z Anesthesia Vaporizer
(Euthanex Corp., Palmer PA, United States). During the surgery,
we made an incision to the midline of the neck, ventral aspect. We
exposed the carotid arteries, dissecting them free from the sheath
and from the vagal nerve as in de la Torre and Fortin (1991).
For the 2VO group, we double-ligated each artery, posterior to
the bifurcation of the carotid, with 4-0 silk sutures. For the sham
group, we dissected the carotids free from the sheath, but did
not proceed with the occlusion. Before closing, we injected each
subject subcutaneously with 1 mg/kg of meloxicam (a surgical
analgesic), as well as either MB or saline. Those subjects randomly
assigned to the MB group were given the first intraperitoneal
injection of MB (4 mg/kg body weight; USP methylene blue,
Spectrum, New Brunswick, NJ, United States). Those subjects in
the saline group received the same volume of physiological saline.
We then sutured the incision and moved the subject to a cage for
recovery, monitoring them for 30 min closely, before returning
the subject to the home cage.
For the following 30 days, each subject in the MB
group received one intraperitoneal injection per day of
4 mg/kg MB, dissolved in saline. The saline group received
intraperitoneal injections of physiological saline vehicle for
30 days. The injections were timed to occur just after each
daily training session, since there is evidence that methylene
blue facilitates memory consolidation when given during the
memory consolidation phase post-training (Martinez et al., 1978;
Callaway et al., 2004). The subjects recovered for 1 week prior to
behavioral testing.
Visual Water Maze Task
Upon recovery from surgery, subjects were trained on the visual
water task (VWT), as described in Prusky et al. (2000). The
VWT using a water Y-maze is a variant of the Morris water
maze, with some additional features. In the visual water Y-maze
task, the subject must make a choice to commit to which arm
Frontiers in Cellular Neuroscience | www.frontiersin.org 3May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 4
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
contains the escape platform. In addition, explicit visual cues
are given to the subject on which arm is correct/incorrect. This
allows for a more complex experimental design than the standard
Morris water maze, one that includes separate sets of “positive”
(+= approach) and “negative” (= avoidance) visual stimuli.
These visual stimuli involve visual patterns that are more similar
to the visual discrimination tests given to humans to test cognitive
function. In paradigms like the elemental discrimination task we
used, these visual cues are progressively added to make the task
progressively more difficult, which would not be possible in the
standard Morris water maze. It is important for this study to
make the maze task progressively more difficult to be able to
reveal progressive memory deficits that develop over days after
chronic 2VO surgery.
A metal water-filled trapezoid-shaped Y-maze tank, with one
end composed of clear Plexiglas, was used for the task as
described in Auchter et al. (2014). Stimuli were presented using
a computer monitor, which was located on the other side of the
transparent wall of the tank. A divider on the midline bisected the
tank, extending from the transparent wall. The divider created
two arms, with two different patterns visible on the monitor at
each end. During each individual trial, a transparent platform
was placed at one arm’s end. It was completely submerged and
invisible to the subject. Each arm was paired with a visual pattern
presented on the monitor. Randomization of the arms for each
trial and recording of behavioral results were controlled by a
computer program (Acumen; CerebralMechanics Inc.). A correct
pattern (+) predicted which arm contained the escape platform.
An incorrect pattern () predicted the arm without the platform.
During the first day of VWT training, the water tank contained
an insert, which was placed such that the rat was forced to
swim down one arm only (without the option of swimming
down the other arm) for 15 trials. The escape platform was
consistently placed at the end of the open arm, and the monitor
was blank (no pattern was presented). The subject was placed
in the tank, and swam around, discovering the platform at the
end. After climbing onto the platform, each rat was removed and
put into a heated recovery cage. Thus, the subjects were trained
to associate swimming and reaching the platform with escape
from the water-filled tank. This also allowed the observation of
swimming ability, independent of the discrimination tasks to
come. All of the subjects successfully learned the location of the
platform at the end of the first few trials. By trial number 10, all
of the subjects were consistently swimming straight to the escape
platform immediately after they were placed in the water.
Visual water task protocols were adapted from those described
by Driscoll et al. (2005). In the first variation of the VWT,
subjects were trained for 10 days on three elemental pattern
discriminations (A+B, C+D, and E+F). Each subject is
placed into the water at the opposite end of the water tank, facing
the monitor. The subject swims into one of the arms, toward
one of the stimuli presented onscreen. If the arm is correct, the
subject climbs onto the escape platform. If the arm is incorrect
(i.e., the escape platform is not present), a metal divider is used
to block that arm for 10 s, then removed. The subject then
swims into the correct arm to reach the escape platform. After
reaching the platform, the subject is removed from the water
and placed in a cage until the next trial begins. Subjects were
trained on 30 trials per day in a stepwise manner: A+Bfirst,
followed by intermixed trials of A+Band C+D, followed by
intermixed trials of A+B, C+D, and E+F. Every subject
reached a criterion of 8/10 correct trials on the A+Bphase
within 4 days. After 10 days, a set of 30 probe trials consisted
of randomly alternated presentations of each of the three sets of
visual stimuli (with 10 trials for each set). In the second variation
of the task, subjects were trained on a more difficult transverse
pattern discrimination (X+Y, Y+Z, and Z+X).
Quantitative Cytochrome Oxidase
Histochemistry
Cytochrome oxidase brain mapping was conducted by an
investigator blind to experimental condition through a well-
established method of optical densitometry of brain sections
as detailed by Gonzalez-Lima and Cada (1994). Following
behavioral procedures, animals were sacrificed, and brains were
quickly removed and frozen in isopentane. Coronal brain
sections (40 µm thick) were obtained and mounted on slides
using a cryostat microtome (Microm HM-505E, Heidelberg,
Germany). To obtain increasing gradients of CO activity, a
frozen brain homogenate paste was sectioned into different tissue
thicknesses (10, 20, 40, 60, and 80 µm) and mounted on separate
slides. CO activity of the homogenate was spectrophotometrically
assessed and used to calculate CO values for the different section
thicknesses. The brain paste sections were used as calibration
standards for each CO staining batch.
Each CO staining batch consisted of representative cryostat
sections (40 µm thick) from two brain levels mounted on
microscope slides for each subject along with two slides with
a set of 5 standards each. Each batch was processed for CO
histochemistry following the method previously described by
Gonzalez-Lima and Jones (1994). Briefly, slides were fixed
for 5 min with 1.5% glutaraldehyde, rinsed three times in
phosphate buffer with sucrose and preincubated in a solution
containing cobalt chloride and DMSO dissolved in Tris buffer.
After rinsing sections with phosphate buffer (pH 7.6; 0.1 M),
they were incubated at 37C for 1 h in the dark and with
continuous stirring in a solution containing diaminobenzidine,
sucrose, cytochrome c and catalase (Sigma-Aldrich, Barcelona,
Spain) dissolved in phosphate buffer (pH 7.6; 0.1 M). The slides
were then dehydrated in increasing concentrations of ethanol,
coverslipped with permount (Merck, Darmstadt, Germany) and
allowed to dry for 48 h.
To obtain CO activity values for regions of interest (ROIs),
a calibration step tablet (Kodak, density range 0.063.05) and
CO-stained slides were placed on a high-precision illuminator
and digitized using a CCD digital microscope camera (Leica
Microsystems DFC450, Wetzlar, Germany). Images were then
analyzed using ImageJ software, which allows the creation of
a logarithmic calibration curve of optical density units as a
function of pixel (gray level) values in each image. Before analysis,
images were corrected for slide and light box artifacts using
background subtraction. For brain structure analyses, 44 ROIs
were located and outlined in each brain hemisphere in each
Frontiers in Cellular Neuroscience | www.frontiersin.org 4May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 5
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
subject by an experimenter blind to experimental conditions
using a rat brain atlas (Paxinos and Watson, 1996; see Table 1
for a complete list of ROIs and abbreviations). The measurement
obtained was an average optical density (OD) value for the
outlined ROI. To maximize the accuracy of OD values, this
procedure was repeated in three adjacent brain sections for
each ROI, and the mean of the three sections in both cerebral
hemispheres was used as the overall mean OD value for that ROI.
Mean OD values were later converted into mean CO activity
units using calibration curves based on tissue standards and
spectrophotometrically determined CO activity. This method
yielded a linear relationship (r>0.95) between biochemical CO
activity measured spectrophotometrically and histochemical CO
reactivity measured by optical density.
Figure 7 shows how the ROI was outlined for CO measures.
For regional CO activity measures, when 2VO-induced lesions
were present within the ROI, both the affected and surviving
tissue were included in the outlined ROI (i.e., the ROI was defined
using the brain atlas, not the lesion). The reason for doing this
was two-fold. First, it allowed us to index general CO activity in
each ROI, taking into account both lesions and compensatory
increases in CO in surviving tissue (if such compensatory
phenomena existed). Second, since some lesions did not have
clearly defined borders, including the entire ROI gave us
measures of CO activity that were unbiased by experimenter
evaluation of lesioned vs. penumbra vs. healthy tissue.
Lesion Volume Analysis
An assessment of lesion volume was conducted in a separate
analysis. In 2VO subjects that showed lesions (n= 6), volumetric
analysis was conducted on affected areas. Lesion volumes (V)
were derived from the lesion area per slice (A) and distance
between collected sections (d), using the formula V = 6A×d.
The value d was calculated as d= (T) ×(number of designated
sections-1), where T is the distance between every designated
section (80 µm). Lesion area per slice was measured by setting
a conservative optical density threshold aimed to prevent the
inclusion of any white matter and tissue artifacts as affected tissue.
This was especially important since we observed lesions in the
striatum, which in rats is speckled with white matter tracts. To
ensure the threshold was unbiased, for each subject the minimum
and maximum threshold values were set to 85% and 98% of the
measured mean OD value for white matter (i.e., “more OD” than
white matter, but “less OD” than healthy gray matter tissue).
A few subjects showed tissue loss where lesions occurred. In these
cases, the maximum value for the threshold was set equal to white
matter so that regions with missing tissue were still considered in
the calculation of lesion area.
Statistical Analyses
All statistical testing was carried out using the PASW 22 software
package (SPSS, Inc., Chicago, IL, United States). All differences
were considered statistically significant at the two-tailed p<0.05
level. Effect sizes were estimated using Cohen’s d (Cohen, 1988).
For univariate group comparisons, average CO values for each
ROI were used as dependent variables in separate analyses, with
surgery and drug treatment as independent variables.
Since subjects went through two iterations of the visual
water task (elemental discrimination and transverse pattern
discrimination), a composite “VWT performance score” was
calculated by averaging the scores from the final probe for
each task. Though subjects did not learn the transverse task
(as indicated by the inability to reach and 8/10 criteria for
pattern discriminations), their performance in the task likely still
influenced CO activity. Thus, brain-behavior correlations were
conducted using two-tailed bivariate correlations between the
VWT performance score and mean CO activity for each ROI.
Interregional correlation of cytochrome oxidase activity was
analyzed in two ways. First, two-tailed bivariate correlations were
performed between all brain ROIs within each experimental
condition. Then, to detect which regional correlations showed
significant differences between groups, correlation coefficients
were compared using Fisher’s z-transformations, followed by
independent samples t-tests (Vélez-Hernández et al., 2014).
RESULTS
The results were obtained by comparisons between four groups
(2 ×2 design): sham +saline group, sham +MB group, 2VO
+saline group, and 2VO +MB group. Since the experimental
design was the same as in Auchter et al. (2014), detailed
explanations of behavioral procedures (and behavioral results)
can be found there, and an experimental timeline is shown in
Figure 1.
Region of Interest (ROI) Analysis
Table 1 shows mean CO values and standard errors for each ROI
in each experimental condition. Only the secondary visual cortex
(V2) showed a significant main effect of surgery, F(1,31) = 4.491,
p= 0.047, with an effect size (Cohen’s d) of 0.82, indicating a
large effect size, given that | d| <0.2 = small effect; 0.2 <| d|
<0.8 = medium effect; | d| >0.8 = large effect (Cohen, 1988).
There were no main effects for drug treatment. Several regions
showed significant surgery by drug treatment interactions,
including perirhinal cortex, F(1,31) = 5.667, p= 0.024, Cohen’s
d = 0.81; secondary motor cortex, F(1,31) = 5.883, p= 0.022,
Cohen’s d = 0.73; primary visual cortex, F(1,31) = 4.606,
p= 0.045, Cohen’s d = 0.74; basolateral amygdala, F(1,31)
= 5.350, p= 0.028, Cohen’s d = 0.61; medial amygdala,
F(1,31) = 5.189, p= 0.030, Cohen’s d = 0.63; and the
anterior portions of the hippocampus, subfields CA1,
F(1,31) = 4.951, p= 0.034, Cohen’s d = 0.48; subfield CA2, F(1,31)
= 4.650, p= 0.039, Cohen’s d = 0.53; and subfield CA3,
F(1,31) = 4.940, p= 0.034, Cohen’s d = 0.53. These significant
surgery by drug treatment interactions are shown in Figure 2.
Interregional Correlation of Cytochrome
Oxidase Activity
To analyze interregional correlation of cytochrome oxidase
activity, CO values for each ROI were correlated with each
other for each group. Functional heat maps for these correlation
matrices are shown in Figures 3,4. Upon visual comparison
of these maps, one can qualitatively deduce that MB treatment
Frontiers in Cellular Neuroscience | www.frontiersin.org 5May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 6
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
TABLE 1 | Mean CO activity values (µmol/min/g) ±standard errors (SEM) for each ROI and each experimental condition.
ROI Sham ±saline SEM Sham ±MB SEM 2VO ±saline SEM 2VO ±MB SEM
Prelimbic cortex (PLC) 246.43 ±9.88 243.80 ±13.63 225.09 ±16.21 247.76 ±9.69
Medial orbital cortex (MO) 245.24 ±11.49 239.99 ±11.85 221.16 ±15.31 248.61 ±9.82
Ventral orbital cortex (VO) 254.38 ±9.57 248.35 ±12.49 235.53 ±12.42 254.43 ±8.99
Lateral orbital cortex (LO) 251.27 ±8.88 249.62 ±11.52 233.50 ±11.32 252.09 ±7.87
Agranular insular cortex (AI) 227.63 ±7.31 220.18 ±8.90 211.33 ±10.82 229.76 ±8.69
Infralimbic cortex (IL) 281.87 ±9.58 270.41 ±14.29 269.97 ±15.16 272.44 ±5.28
Primary motor cortex (M1) 237.17 ±4.92 230.15 ±10.02 222.54 ±13.23 232.86 ±3.74
Secondary motor cortex (M2) 242.28 ±6.10 233.62 ±10.50 214.49 ±8.99 243.12 ±7.61
Primary somatosensory cortex (S1) 214.27 ±13.65 214.25 ±7.76 222.05 ±11.91 212.94 ±7.10
Secondary somatosensory cortex (S2) 226.35 ±7.55 218.51 ±10.34 210.28 ±7.41 230.64 ±8.22
Cingulate cortex (Cin) 259.93 ±8.32 244.58 ±10.16 258.77 ±15.84 257.88 ±9.85
Perirhinal cortex (PRh) 230.05 ±10.87 210.98 ±8.89 186.46 ±14.63 223.64 ±10.99
Primary visual cortex (V1) 241.59 ±10.36 242.43 ±7.87 201.47 ±12.24 247.19 ±9.66
Secondary visual cortex (V2) 249.72 ±11.05 249.49 ±9.86 206.10 ±13.52 239.80 ±7.90
Medial septal nucleus (MS) 203.40 ±8.33 197.97 ±12.39 202.64 ±10.36 197.97 ±6.04
Lateral septal nucleus (LS) 272.30 ±8.28 262.01 ±12.81 273.92 ±11.81 268.67 ±5.58
Acumbens core (AcbC) 313.40 ±9.76 304.24 ±10.43 315.15 ±17.36 307.88 ±7.59
Acumbens shell (AcbS) 241.13 ±10.53 223.67 ±8.36 233.98 ±8.35 224.90 ±7.96
Caudate/Putamen (CPu) 198.87 ±13.32 199.51 ±8.52 191.14 ±11.26 191.27 ±6.12
Globus palidus (GP) 115.97 ±6.57 108.53 ±4.83 115.23 ±5.74 112.64 ±4.92
Ventral pallidum (VP) 179.11 ±5.82 169.26 ±6.40 168.38 ±8.74 168.31 ±4.50
Lateral hypothalamus (LH) 160.75 ±9.32 157.53 ±9.78 154.51 ±5.90 163.81 ±8.06
Paraventricular nucleus (PVH) 191.74 ±6.94 183.50 ±8.14 184.33 ±8.43 203.21 ±6.86
Medial preoptic area (MPO) 206.22 ±6.62 198.81 ±7.51 210.35 ±10.84 208.42 ±5.72
Bed nucleus stria terminalis (BST) 233.80 ±7.98 218.64 ±9.49 237.61 ±14.51 224.12 ±6.61
Subthalamic nucleus (Sub) 273.20 ±6.04 249.10 ±10.74 249.36 ±14.37 248.75 ±10.91
Medial geniculate nucleus (MGN) 255.28 ±8.38 245.48 ±5.66 240.78 ±12.86 235.70 ±5.45
Basomedial amygdala (BMA) 186.21 ±7.73 180.59 ±10.02 175.09 ±4.88 195.70 ±9.16
Medial amygdala (MEA) 227.95 ±10.39 220.31 ±11.47 194.29 ±9.88 226.00 ±9.75
Basolateral amygdala (BLA) 268.46 ±14.51 254.36 ±14.44 230.77 ±12.21 266.47 ±12.53
Central amygdala (CEA) 223.13 ±8.63 219.37 ±10.03 206.30 ±9.63 234.55 ±9.36
Anterior hippocampus CA1 (CA1A) 213.48 ±11.42 199.98 ±8.20 188.32 ±18.91 213.97 ±13.79
Anterior hippocampus CA2 (CA2A) 224.26 ±13.21 203.44 ±8.69 195.70 ±16.03 214.87 ±11.19
Anterior hippocampus CA3 (CA3A) 238.88 ±15.71 213.68 ±10.05 200.94 ±15.59 216.38 ±8.13
Anterior dentate gyrus (DGA) 319.76 ±26.01 294.90 ±15.21 306.25 ±31.40 297.03 ±19.93
Posterior hippocampus CA1 (CA1P) 298.13 ±13.98 277.27 ±12.72 266.56 ±14.28 277.08 ±13.51
Posterior hippocampus CA2 (CA2P) 275.84 ±11.87 249.69 ±11.82 236.40 ±17.96 248.44 ±10.82
Posterior hippocampus CA3 (CA3P) 292.89 ±9.91 272.22 ±13.02 255.42 ±16.45 269.08 ±9.99
Posterior dentate gyrus (DGP) 269.60 ±15.75 256.92 ±10.66 258.72 ±12.20 255.88 ±7.02
Subiculum (S) 239.98 ±12.04 226.65 ±10.53 221.74 ±11.20 217.73 ±6.72
Superior colliculus (SC) 254.18 ±13.53 246.46 ±8.80 240.89 ±10.46 249.47 ±8.05
Substantia nigra (SN) 214.80 ±8.68 207.29 ±8.24 211.66 ±11.57 209.81 ±6.72
Red nucleus (R) 250.37 ±12.02 251.90 ±13.05 234.69 ±13.86 257.29 ±12.34
Ventral tegmental area (VTA) 97.29 ±1.32 99.91 ±5.86 88.06 ±3.55 96.05 ±5.11
Means showing an individual group effect are boldfaced.
generally increased interregional correlation of cytochrome
oxidase activity, specifically in a positive direction (i.e., MB
resulted in more “redness” in the heat maps). This is true
particularly in the sham condition (Figure 3). The result
of MB treatment in the 2VO condition is not as obvious,
though still apparent (Figure 4). One can also compare the
left sides of Figures 3,4to observe an apparent decrease
in interregional correlation of cytochrome oxidase activity as
a result of 2VO surgery. The most dramatic differences in
interregional correlation of cytochrome oxidase activity appear in
the outlined boxes in Figures 3,4(also represented in larger form
in Figure 5), which represent connectivity between prefrontal
and visual cortex ROIs, and amygdala and hippocampal ROIs.
Figure 5 shows stronger interregional correlation of cytochrome
Frontiers in Cellular Neuroscience | www.frontiersin.org 6May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 7
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
FIGURE 2 | Regions of interests showing significant effects. In these regions, 2VO surgery (red) resulted in a decrease in cytochrome oxidase activity, which was
prevented by MB treatment (violet). M2, secondary motor cortex; V1, primary visual cortex; V2, secondary visual cortex; BLA, basolateral amygdala; MEA, medial
amygdala; PRh, perirhinal cortex; CA1A, anterior hippocampus CA1; CA2A, anterior hippocampus CA2; CA3A, anterior hippocampus CA3. *Significant surgery by
drug interaction (p<0.05); ˆSignificant main effect of surgery (p<0.05).
oxidase activity in the sham animals given MB as compared
to saline. Importantly, it appears that the connectivity between
cortical, amygdala and hippocampal ROIs is decreased in subjects
that received 2VO, but connectivity is restored in 2VO subjects
treated with MB.
To further explore the effects of cerebral hypoperfusion
and MB treatment on interregional correlation of cytochrome
oxidase activity, bivariate correlation comparisons for each
ROI were conducted (1) between the sham +saline group
(control subjects) and 2VO +saline group, and (2) between
the 2VO +saline group and the 2VO +MB group (Figure 6).
The first group of comparisons essentially reveals which
interregional correlations were significantly strengthened or
weakened as a result of cerebral hypoperfusion. The second
group of comparisons reveals which interregional correlations
were significantly strengthened or weakened as a result of MB
treatment during cerebral hypoperfusion.
As shown in the left panel of Figure 6, interregional
correlation of cytochrome oxidase activity between visual cortices
and basal forebrain, hippocampal and midbrain ROIs was
weakened as a result of cerebral hypoperfusion. However,
interregional correlation of cytochrome oxidase activity between
cortical regions—particularly between secondary somatosensory
cortex and prefrontal ROIs—was strengthened as a result of
cerebral hypoperfusion. This could potentially be evidence of a
compensatory mechanism activated in response to the functional
disconnection between regions involved in the visual network.
As shown in the right panel of Figure 6, interregional
correlation of cytochrome oxidase activity between visual cortices
and hippocampal ROIs—which was weakened in 2VO subjects—
was strengthened as a result of MB treatment. Additionally,
MB treatment resulted in increased interregional correlation of
cytochrome oxidase activity between amygdala and cortical ROIs,
and in midbrain ROIs with visual cortices and hippocampus.
Neurodegenerative Lesions
Figure 7 illustrates the procedure for obtaining a conservative
measure of neurodegenerative lesion area per section. About
one third of subjects who received 2VO surgery (n= 3/9
in the 2VO +saline group and n= 3/8 in the 2VO +
MB group) showed conspicuous anatomical lesions (Figure 8).
Though there was quite a bit of heterogeneity in lesion size
between subjects, the location of the lesions were categorically
delimited, and thus were classified into 4 types: neocortical
(occurring unilaterally or bilaterally in prefrontal regions and
extending into primary motor, somatosensory and visual cortices;
n= 2), striatal (occurring unilaterally in the caudate and
putamen; n= 6), hippocampal (occurring unilaterally through
the rostrocaudal extent of CA1, CA2 and CA3 subfields;
n= 2), and perirhinal (occurring unilaterally or bilaterally in
Frontiers in Cellular Neuroscience | www.frontiersin.org 7May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 8
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
FIGURE 3 | Heat maps showing interregional correlations for the sham + saline group (left) and sham + MB group (right). The purpose of these diagrams is to
show the differences in the overall pattern of correlation, rather than individual pairs. Colors correspond to Pearson’s r correlation coefficients, with red indicating
strong positive correlation and blue indicating strong negative correlations. Generally, the MB group showed a greater number of positive correlations (red) than the
saline group. Black boxes outline correlations between prefrontal ROIs and amygdala ROIs and prefrontal ROIs and hippocampal ROIs.
FIGURE 4 | Heat maps showing interregional correlations for the 2VO + saline group (left) and 2VO + MB group (right). Colors correspond to Pearson’s r
correlation coefficients, with red indicating strong positive correlations and blue indicating strong negative correlations. Black boxes outline correlations between
prefrontal ROIs and amygdala ROIs and prefrontal ROIs and hippocampal ROIs.
perirhinal cortex; n= 3). Qualitative and quantitative lesion
comparisons revealed several interesting phenomena. First, there
was a negative linear correlation between lesion size and VWT
performance, indicating that the greater the overall lesion
volume, the worse subjects performed on the VWT (Figure 8).
Second, though the number of subjects displaying lesions is
Frontiers in Cellular Neuroscience | www.frontiersin.org 8May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 9
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
FIGURE 5 | Heat map comparing interregional correlation of cytochrome oxidase activity between prefrontal ROIs and amygdala and hippocampal ROIs in each
experimental condition. Colors correspond to Pearson’s r correlation coefficients, with red indicating strong positive correlations and blue indicating strong negative
correlations.
FIGURE 6 | Significant differences in interregional correlation of cytochrome oxidase activity due to 2VO surgery and MB treatment. (A) Comparison between control
subjects (sham + saline group) and 2VO subjects (2VO + saline). 2VO surgery resulted in weaker interregional correlation of cytochrome oxidase activity (1) within
ROIs involved in the visual pathway (V1, V2, SC), and (2) between visual ROIs and hippocampus (CA3, DG, S). Stronger interregional interregional correlation of
cytochrome oxidase activity was observed within cortical ROIs (MO, VO, PLC, IL, S1, Cg). (B) Comparison between 2VO subjects treated with saline and those
treated with MB. In 2VO subjects, treatment with MB resulted in stronger interregional correlation of cytochrome oxidase activity (1) within ROIs involved in the visual
pathway (V1, V2, SC), (2) between visual ROIs and hippocampus (CA1, CA2, CA3, DG, S), (3) between cortical ROIs (LO, VO, AI, M1) and hippocampus (CA1, CA2,
DG), and (4) between cortical ROIs (VO, PLC, M1) and amygdala (BMA, CeA). Mildly weaker interregional correlation of cytochrome oxidase activity was observed
between the amygdala (CeA), hippocampus (DG), and striatum (GP).
Frontiers in Cellular Neuroscience | www.frontiersin.org 9May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 10
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
FIGURE 7 | (A) Delineation of region of interest for CO measurement. For regional CO activity measures, when 2VO-induced lesions were present within the ROI,
both the affected and surviving tissue were included in the outlined ROI (i.e., the ROI was defined using the brain atlas, not the lesion). (B) Determination of
2VO-induced lesion volume in rat brain. For every coronal section showing a lesion (e.g., lateral side of striatum), a threshold was applied to highlight (in red) tissue
with optical density (OD) values between that of white matter and healthy gray matter tissue (medial side of striatum). For each section showing a lesion, the area of
the highlighted tissue within the approximate lesion outline (in yellow) was used as A in the equation to calculate lesion volume, V = 6A×d, where d is the distance
between sections. The sections were stained for CO activity by the quantitative enzyme histochemistry method of Gonzalez-Lima and Jones (1994). We are showing
the actual digitized images we used for the densitometric quantification of CO activity. Microphotographs with high contrast are not suitable for demonstration of a
linear staining reaction product resulting from graded CO enzymatic activity as explained in our book on quantitative CO enzyme histochemistry with more details on
this topic (Gonzalez-Lima, 1998). Briefly, digitized images with low contrast showing a linear gray level range are necessary to perform densitometric computer
analysis of quantitative differences in CO activity from histochemically stained frozen brain sections and CO activity standards.
FIGURE 8 | Six 2VO subjects showed neurodegenerative lesions (n= 3 saline-treated [pink background], n= 3 MB-treated [purple background]). There was a
negative linear correlation between lesion size and VWT performance (top left). We are showing actual examples of digitized images of CO-stained coronal sections
used for the determination of lesion volume.
Frontiers in Cellular Neuroscience | www.frontiersin.org 10 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 11
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
small, it appears that the emergence of the lesions followed a
specific pattern. In subjects with the smallest lesion volumes,
lesions were limited to striatum. In more severe cases, neocortical
and perirhinal lesions appeared (Figure 8). Subjects with the
largest lesion volumes showed striatal, neocortical, perirhinal
and hippocampal lesions. Finally, though both carotid arteries
were ligated in the 2VO surgery, when subjects had smaller
lesions, the lesions were limited to either the right or the
left cerebral hemisphere. Bilateral lesions only appeared in the
most severe cases (in which subjects showed lesions in multiple
locations, including hippocampus). Perhaps the most interesting
observation in subjects displaying lesions was the impact of MB
treatment. Though the same number of subjects showed lesions
in each 2VO group, lesions in subjects treated with MB were
smaller and more localized (i.e., only unilateral and only in/near
striatum) than lesions in saline-treated 2VO subjects. The lesion
volumes for each of the six subjects (as well as treatment group
means) are shown in Figure 9.
BrainBehavior Correlations
Like the behavior performance for the elemental discrimination
alone (Auchter et al., 2014), a univariate ANOVA—with the
composite VWT performance score as the dependent variable
and the surgery and drug treatment conditions as independent
variables—revealed a surgery by drug treatment interaction,
F(1,31) = 8.961, p= 0.006. Post hoc pairwise comparisons
demonstrated that subjects who received 2VO surgery performed
worse than sham subjects (decreased percent correct responses),
but this was ameliorated by MB treatment (Figure 10).
Visual water task performance scores were correlated with CO
activity for a number of individual ROIs. Regions that showed
significant positive brain-behavior correlations were perirhinal
cortex, r(35) = 0.526, p= 0.001, prelimbic cortex, r(35) = 0.353,
p= 0.038, secondary motor cortex, r(35) = 0.461, p= 0.005,
primary visual cortex, r(35) = 0.432, p= 0.035, secondary visual
cortex, r(35) = 0.410, p= 0.047, caudate/putamen r(35) = 0.343,
p= 0.043, medial amygdala, r(35) = 0.368, p= 0.030, anterior
CA1, r(35) = 0.455, p= 0.006, anterior CA2, r(35) = 0.390,
p= 0.021, and anterior CA3, r(35) = 0.435, p= 0.009. There
were no significant negative correlations between CO activity and
VWT performance.
To assess whether correlations between regional CO activities
and VWT performance were specifically driven by subjects
showing lesions, brain-behavior correlations were also conducted
excluding subjects who displayed lesions. When lesioned subjects
were excluded, perirhinal cortex, r(29) = 0.397, p= 0.033,
secondary motor cortex, r(29) = 0.409, p= 0.027, medial
amygdala, r(29) = 0.424, p= 0.022, primary visual cortex,
r(29) = 0.533, p= 0.0323, and secondary visual cortex,
r(29) = 0.471, p= 0.049 still showed significant positive
correlations between CO activity and behavioral performance.
Hippocampal regions, prelimbic cortex and caudate/putamen no
longer showed significant correlations. Interestingly, significant
correlations emerged in agranular insular cortex, r(29) = 0.392,
p= 0.035, and lateral orbital cortex, r(29) = 0.435, p= 0.018, when
lesioned subjects were excluded. That many correlations persisted
when lesions were absent suggests that the lesions are not
FIGURE 9 | Lesion volume (cubic centimeters) in each of the six 2VO subjects
which showed neurodegenerative lesions. Lesions were observed in n= 3 MB
group subjects and n= 3 saline group subjects. Methylene blue treatment
after 2VO surgery reduced the size of the lesions. Bars on means are
standard errors.
FIGURE 10 | Visual water task performance scores. 2VO surgery resulted in
worse overall scores on the VWT. However, 2VO subjects treated with MB
performed no different from sham groups; *p<0.05.
entirely responsible for the observed brain-behavior correlations.
However, correlations were not completely independent of
lesions either, since some correlations disappeared when lesioned
subjects were excluded. Further, since additional correlations
appeared in non-lesions subjects (in ROIs that did not show
lesions in lesioned subjects), the functional impact of lesions
extended beyond the lesioned areas themselves.
DISCUSSION
Brain cells rely on the circulatory system to supply the necessary
glucose and oxygen needed for mitochondrial respiration,
oxidative phosphorylation and ATP synthesis (Erecinska and
Frontiers in Cellular Neuroscience | www.frontiersin.org 11 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 12
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
Silver, 1989). Cytochrome oxidase (CO)a transmembrane
protein localized in the inner mitochondrial membrane—is the
terminal enzyme of the electron transport chain (Wong-Riley,
1989). It is well-known that chronic cerebral hypoperfusion
results in impaired ATP synthesis (Plaschke, 2005;Br¯
ıede and
Duburs, 2007), mitochondrial dysfunction and a reduction in
CO activity (de la Torre et al., 1997;Cada et al., 2000).
There is also evidence that this mitochondrial dysfunction is
paralleled by deficits in learning and memory (de la Torre et al.,
1997;Cada et al., 2000). Here, we used a model of chronic
cerebral hypoperfusion, bilateral carotid occlusion (2VO), the
neuropathological effects of which have been well-characterized
and resemble those of age-related progression of mild cognitive
impairment (MCI) and Alzheimer’s disease (AD) (de la Torre,
2016). We aimed to: (1) characterize the whole-brain regional and
interregional correlation of cytochrome oxidase activity changes
that result from 2VO surgery, (2) describe how these changes
are ameliorated via treatment with the neurometabolic enhancer
USP methylene blue (MB), and (3) correlate brain CO activity
with performance on the visual water task (VWT).
Regional Changes in Cytochrome
Oxidase Activity
Regional changes in cytochrome oxidase activity as revealed by
univariate analyses for each ROI were observed in cortical regions
(secondary motor cortex, perirhinal cortex, primary visual cortex
and secondary visual cortex), amygdala (basolateral and medial)
and hippocampus (the anterior portions of CA1, CA2, and CA3).
Though the neuropathological changes in hippocampus (Shang
et al., 2005), retina (Lavinsky et al., 2006), and optic tract (Farkas
et al., 2004) as a result of 2VO have been documented, regional
differences in cortical areas specific to visually guided movements
have only been documented one other time (Cada et al., 2000);
and regional differences in the amygdala are novel findings.
Additionally, we show here that chronic cerebral
hypoperfusion can affect regional cytochrome oxidase activity
in two ways. First, regions susceptible to hypoperfusion show
decreases in CO activity without developing anatomical lesions.
This is evidenced by the fact that we observed univariate
differences in CO activity as a result of 2VO surgery in regions
that did not show lesions (e.g., in basolateral and medial
amygdala). Interregional correlation of cytochrome oxidase
activity changes observed in response to 2VO surgery in regions
that do not show anatomical lesions further support this notion.
Second, in about one third of subjects, 2VO surgery resulted in
lesions, as indicated by absence of histochemical CO staining and
presence of tissue damage. Studies comparing the CO method
and other histological methods show that CO activity is a good
proxy for cell density (Nelson and Silverstein, 1994;Wong-Riley
and Welt, 1980;Riha et al., 2005). Thus we can infer that regions
showing complete absence of CO activity in discrete identifiable
regions are in fact damaged. Since this happens in less than
half of the subjects and that lesion sizes vary so widely suggests
that subjects are differentially susceptible to developing lesions
during cerebral hypoperfusion after 2VO. This differential
susceptibility not only results in variable lesion sizes, but greater
lesion sizes are also highly correlated to impaired behavioral
performance in the VWT.
Additionally, that lesions appear in regions where CO activity
is particularly involved in visual discrimination learning (Fidalgo
et al., 2014)and that CO activity in these regions were also
correlated with VWT performancebrings about the question of
whether the lesions caused the behavioral deficits, or whether the
intensive behavioral training facilitated the lesions. In one likely
scenario, we see the appearance of lesions as a result of certain
regions being particularly vulnerable to cerebral hypoperfusion.
The lesions would then lead to an inability of these subjects to
master the VWT task. However, in an alternative scenario, the
VWT itself may have resulted in increased CO activity demand
in particular regions preceding neurodegeneration. In this case,
cerebral hypoperfusion could result in increased oxidative stress
in regions involved in learning the VWT task, and thus would
result in “customized” lesions in brain regions involved in a
particular task. Though both scenarios may have contributed in
some way to the appearance of lesions, we believe that the former
scenario is the most plausible, primarily because similar regions
have been previously identified as susceptible to cerebrovascular
ischemia. Additionally, since CO activity in the medial amygdala
is correlated with VWT performance, if lesions resulted from
the demanding VWT training, then we should also expect to see
amygdala lesions, which we did not. The potential emergence
of task-specific lesions can be further addressed in future
investigations by (1) comparing mitochondrial lesions in trained
vs. untrained subjects experiencing cerebral hypoperfusion,
and/or (2) training subjects on a different task involving different
brain regions (e.g., an auditory rather than visual task), and
exploring whether the lesion location changes (e.g., to auditory
rather than visual cortices).
Interregional Correlations of Cytochrome
Oxidase Activity
2-vessel occlusion surgery resulted in weakened interregional
correlation of cytochrome oxidase activity in several regions.
The most obvious decoupling occurred between visual cortices
(primary and secondary) and hippocampus (CA3, dentate and
subiculum). 2VO subjects also showed weaker interregional
correlation of cytochrome oxidase activity between visual cortex
and the superior colliculus, further implicating 2VO surgery
in the pathogenesis of the visual system. There were also
some noteworthy increases in interregional correlation of
cytochrome oxidase activity, namely between the subthalamic
nucleus and prefrontal cortex (ventral and medial orbital
cortex), between the bed nucleus of the stria terminalis and
nucleus accumbens, and between several cortical regions (medial
orbital cortex, prelimbic cortex, infralimbic cortex, cingulate,
and primary somatosensory cortex). Because these increases
in interregional correlation of cytochrome oxidase activity
seemed to appear mostly in regions that were not significantly
affected by 2VO surgery (as indicated by univariate analyses),
this strengthened interregional correlation of cytochrome
oxidase activity may indicate a compensatory mechanism that
occurred in response to the weakened interregional correlation
Frontiers in Cellular Neuroscience | www.frontiersin.org 12 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 13
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
of cytochrome oxidase activity in visual and hippocampal
regions. There is some evidence of compensatory mechanisms
in 2VO, however most of this evidence concerns global
normalization of cerebral blood flow via vascular changes,
such as collateral perfusion and angiogenesis (Choy et al.,
2006). Therefore, there is the possibility that MB may have
preserved CO activity and memory by partly alleviating
hypoperfusion in the ischemic brain, e.g., by increasing blood
supply via the vertebral arteries after occluding the carotid
arteries. Our own fMRI studies of cerebral blood flow indicate
that MB can enhance blood flow under hypoxic conditions
(Huang et al., 2013). To verify the existence of compensatory
mechanisms in blood flow and metabolic activity, further
investigation is needed.
Biological Underpinnings of the
Interregional Correlations of Cytochrome
Oxidase Activity
At the cellular level, CO enzymatic activity reflects neuronal
synaptic activity because CO is a rate-limiting enzyme in
cellular respiration for ATP production, and ATP is required
to sustain neuronal electrophysiological responses (Wong-
Riley, 1989). Specifically, neuronal membranes depolarized by
synaptic excitatory transmitters require ATP for repolarization
to maintain electrophysiological responses. Moreover, at the
transcriptional molecular level, there is a coupling of genes for
CO enzymatic activity with genes for neuronal synaptic excitation
(Wong-Riley, 2012). Specifically, the transcriptional regulation of
CO genes is coupled with the transcription of nuclear respiratory
factor genes, such as NRF-1 and NRF-2, which in turn are coupled
with transcription of excitatory neurotransmitter receptors, such
as NMDA receptor subunit genes for glutamatergic excitatory
synaptic activity (GluN1 and GluN2) (Dhar and Wong-Riley,
2009;Wong-Riley, 2012;Nair and Wong-Riley, 2016). Therefore,
all ten nuclear CO genes and the three mitochondrial CO
transcription factors are transcribed in the same “transcription
factory” that is neuronal activity-dependent (Wong-Riley, 2012).
This makes quantitative CO histochemistry of individual brain
regions an important metabolic index of neuronal activity
(Gonzalez-Lima, 1998). Specifically, CO histochemical activity
after 2VO provides insight into the metabolic history of brain
regions during chronic brain hypoperfusion, as brain regions that
are progressively less active will show reduced CO activity relative
to regions with preserved neuronal activity (Cada et al., 2000).
Metabolic mapping techniques like quantitative CO
histochemistry make it possible to gather functional data
from most brain regions. However, most analyses of metabolic
mapping data are limited to the comparison of mean regional
activity between groups. This is like treating each brain
region as if it were separate from the rest of the brain.
However, the interactions between regions likely affect higher-
order neurobiological functions underlying neural network
communications and associative learning functions (Gonzalez-
Lima and McIntosh, 1995). Differences in regional mean CO
activity after chronic 2VO reflect differences in the cumulative
neuronal activity of specific brain regions, whereas differences
in interregional correlations of CO activity reflect cumulative
differences in functional synaptic coupling among brain regions.
At the interregional physiological level, CO activity is critical for
the coordinated functioning of brain regions because aerobic
cellular respiration is the main way neurons obtain metabolic
energy to communicate via synapses. Hence, interregional
correlations of CO activity represent a functional connectivity
index of the metabolic history of interactive neuronal synaptic
activity among brain regions (Gonzalez-Lima, 1998). However,
the biological underpinnings of CO interregional correlations
are very different from other metabolic markers such as
uptake of 2-deoxyglucose or fluorodeoxyglucose, or c-fos
gene/protein expression, all of which reflect evoked or immediate
neuronal activity. As explained in detail by Sakata et al. (2000),
interregional correlations of CO activity represent functional
“traits” rather than an acute “state.”
In our study, differences in the magnitude of interregional CO
correlations after chronic 2VO and MB reflected the metabolic
history of functional synaptic relationships between brain regions
that were part of specific neural networks. Although interregional
CO effects need to happen via anatomical synaptic connectivity,
anatomical and functional connectivity are not synonymous. For
example, two regions may be similarly connected anatomically
in the saline groups and the MB groups, but differences in their
interactive synaptic activity would lead to different interregional
correlations of CO activity. Specifically, we found different
interregional correlations in CO activity among prefrontal and
limbic brain regions within groups of subjects treated with
saline or MB from sham and 2VO groups. We then established
the patterns of statistical significance for each condition of
treatment (saline/MB) and surgical (sham/2VO) groups and
interpreted these results as differences in the metabolic history
of functional synaptic connections between brain regions due
to each condition.
Horwitz et al. (1992) reviewed the first studies that
introduced the analysis of functional network interactions using
interregional correlations of metabolic mapping data in the
1980s. This network approach became popular to analyze the
relationships of blood oxygen level dependent (BOLD) signals
from multiple brain regions in fMRI, and Friston (1994) called
it functional connectivity analysis. The only difference in these
analytic techniques is the type of metabolic data used. However,
all these network computational techniques are based on the
same assumption that brain regions that function together
have correlated activities. Gonzalez-Lima and McIntosh (1994)
explicitly stated this assumption as the principle of neural
interaction: “It states that if neural regions are synaptically
connected, the disturbance in the postsynaptic action potentials
of a region is passed on to another. In other words, brain
regions do not merely act locally, they interact with one
another in complex neural networks. Therefore, brain activity is
interdependent on the actions and reactions of the components
that form the neural networks.” (Gonzalez-Lima and McIntosh,
1994, p. 24).
Interpreted in terms of neural network analysis, an
interregional correlation of CO activity represents the degree to
which the neuronal synaptic activity between two regions are
Frontiers in Cellular Neuroscience | www.frontiersin.org 13 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 14
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
related to one another, or how they vary together (covariance).
As explained by McIntosh and Gonzalez-Lima (1994), a high
interregional correlation between regions A and B means that
if region A increases its activity, so too will B, in the case of a
positive correlation. The brain is unique among other organs in
that it is made of interconnected elements critically dependent
on CO activity for synaptic communication, from the local
synaptic activity of neurons, to interregional connections among
ensembles of neurons across brain regions. Communication
among neural elements, whether neurons or ensembles, is along
these interconnections, and these network communications
represent a large-scale biological underpinning of brain function.
In other words, a change in interregional CO activity correlations
between neural regions comes about through a change in the
synaptic influences of one or more input pathways. Therefore,
we identified changes in network interactions after 2VO and
MB interventions by examining the interregional correlations
of CO activity within regions of the brain. As brain regions
became progressively less active after 2VO, their synaptic
communications became weaker, resulting in a reduced number
of significant interregional correlations. As MB facilitated
CO activity in brain regions, so the interregional synaptic
communications were stronger, resulting in a higher number of
significant interregional correlations.
Methylene Blue Preserves Cytochrome
Oxidase Activity and Prevents
Neurodegeneration in Chronic Cerebral
Hypoperfusion
The enzymatic activity of CO is responsible for the consumption
of oxygen by catalyzing the reaction that reduces oxygen into
water in the process of oxidative phosphorylation that generates
ATP (Erecinska and Silver, 1989). By preserving CO activity
MB can improve oxygen consumption even under hypoxic
conditions. For example, Huang et al. (2013) demonstrated that
MB has a stronger effect under mild hypoxic conditions by
comparing normoxia and hypoxia MB effects in vivo. MB under
hypoxia induced greater stimulus-evoked fMRI responses and
oxygen consumption as compared to normoxia. They concluded
“Such enhanced potentiation during hypoxia could be one of
the mechanisms that accounts for MB’s neuroprotective effects
in metabolically stressed conditions reported in the literature
(see review Rojas et al., 2012). For example, the higher the
metabolic demand for oxygen consumption, the higher the
respiratory chain electron flow produced by MB’s electron cycling
action in mitochondria (Rojas et al., 2012). Therefore, MB’s
effects during hypoxia may potentiate fMRI responses by further
increasing mitochondrial electron transport. We predict that
more severe (i.e., 9–12% O2) hypoxia could evoke a larger MB
effect.” Furthermore, MB has been shown to protect against brain
injury after cardiac arrest, which produces hypoxia-reperfusion
injury demonstrated by a decrease in the plasma level of
protein S-100Beta, an astroglial marker of hypoxic brain injury
(Miclescu et al., 2006).
While it is outside the scope of this paper to provide a
comprehensive review of all the properties and effects of MB,
we refer the reader to a recent paper that reviews ways in
which MB may be neuroprotective by preserving mitochondrial
function (Tucker et al., 2018). Similarly, more recently other
MB studies have also shown MB’s protective action in multiple
models and tissues. For example, Tian et al. (2018) showed
that MB protects the lungs from ischemia-reperfusion injury
by attenuating mitochondrial oxidative damage. Bhurtel et al.
(2018) showed that MB protects dopaminergic neurons against
MPTP-induced neurotoxicity by up-regulating brain-derived
neurotrophic factor and Biju et al. (2018) showed that MB
reduces motor deficits and olfactory dysfunction in a chronic
MPTP-probenecid mouse model of Parkinson’s disease. Relevant
to our study, Huang et al. (2018) investigated chronic oral MB
treatment in a rat model of focal cerebral ischemia-reperfusion.
However, the present study is the first to investigate MB
neuroprotective effects on mitochondrial CO activity in the
pathogenesis of chronic brain hypoperfusion.
When administered during chronic cerebral hypoperfusion,
MB preserved mitochondrial CO activity in affected brain regions
(secondary motor cortex, perirhinal cortex, primary visual cortex,
basolateral amygdala, medial amygdala and hippocampus). This
effect was so robust that only one region (secondary visual
cortex) that showed decreased CO activity as a result of 2VO was
not restored by MB treatment. Additionally, though the same
number of subjects showed lesions as a result of 2VO surgery,
subjects treated with MB displayed smaller, more localized lesions
than subjects treated with saline.
One potential explanation for the appearance of lesions in
some subjects and not in others is the induction of necrotic
vs. apoptotic mechanisms in response to chronic cerebral
hypoperfusion. Necrosis is a form of neuronal cell injury caused
by external cellular factors (e.g., toxins or trauma) that result
in autolysis, unregulated and detrimental digestion of cellular
components. Apoptosis is programmed or targeted cell death,
the results of which can potentially benefit the organism.
A key determinant of necrosis vs. apoptosis is the presence of
intracellular ATP. In the weeks following 2VO surgery, there is
a rapid depletion of ATP (Plaschke, 2005;Br¯
ıede and Duburs,
2007). This may lead to an induction of necrotic mechanisms in
cells more vulnerable to ATP depletion. If this is indeed the case,
then increases in ATP production as a result of MB treatment
may explain why lesions were smaller and more localized in
MB-treated subjects.
Methylene blue treatment in 2VO surgery also resulted in
stronger interregional correlation of cytochrome oxidase activity
between the same regions that were weakened in saline-treated
subjects (i.e., between visual cortex and hippocampus and visual
cortex and midbrain). MB treatment in sham and 2VO groups
also resulted in widespread strengthened connectivity between
hippocampus (CA1, CA2 and dentate) and prefrontal cortex
(lateral orbital cortex, ventral orbital cortex and anterior insular
cortex), as well as between amygdala (central and medial) and
frontal cortex areas (ventral orbital cortex, prelimbic cortex and
primary motor cortex). Widespread modulation of interregional
correlation of cytochrome oxidase activity by MB has also been
reported in the brains of healthy humans analyzed with fMRI
(Rodriguez et al., 2017).
Though amygdala-cortical interregional correlation of
cytochrome oxidase activity was not significantly affected by
Frontiers in Cellular Neuroscience | www.frontiersin.org 14 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 15
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
2VO surgery, the strengthened interregional correlation of
cytochrome oxidase activity between these regions likely assisted
in the learning of the VWT. The amygdala has a modulatory
role in both spatial and cued water maze tasks (Packard et al.,
1994). The medial amygdala was also positively correlated
with VWT performance, strengthening the likelihood that
the increased amygdala-cortical interregional correlation of
cytochrome oxidase activity we saw as a result of MB treatment
was related to learning the VWT escape task.
It has been demonstrated before that low doses (14 mg/kg)
of methylene blue (1) enhance cytochrome oxidase activity, (2)
are neuroprotective, and (3) enhance learning and memory in
animals and humans (Rojas et al., 2012;Telch et al., 2014;
Echevarria et al., 2016;Rodriguez et al., 2016;Auchter et al.,
2017;Zoellner et al., 2017). However, this is the first study to
integrate all three of these implications to show that MB is
neuroprotective by enhancing mitochondrial activity in regions
specifically vulnerable to hypoperfusion. Additionally, functional
networks that were specifically weakened by 2VO surgery were
restored by daily MB treatment.
CONCLUSION
This is the first study to characterize the functional changes
in brain metabolic activity that result from chronic cerebral
hypoperfusion, and show how treatment with MB ameliorates
these changes, preventing neurodegeneration and memory
impairment. Interregional correlation of cytochrome oxidase
activity was weakened between regions specifically involved
in visual discrimination learning, which likely resulted in
decreased performance in the visual water task. Treatment with
MB not only restored task-specific interregional correlation
of cytochrome oxidase activity, but also restored average
mitochondrial activity in regions specifically affected by 2VO
surgery. These findings implicate MB as a potential prophylactic
or therapeutic treatment for chronic cerebral hypoperfusion,
including people at a high risk for or symptomatic of
neurodegenerative disorders whose pathology is accompanied by
cardiovascular disease or cerebrovascular hypoxia.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation, to any
qualified researcher.
ETHICS STATEMENT
This study was carried out in accordance with the
recommendations of NIH Guide for the Care and Use of
Laboratory Animals, Committee for the Update of the Guide
for the Care and Use of Laboratory Animals. The protocol was
approved by the Institutional Animal Care and Use Committee,
University of Texas at Austin.
AUTHOR CONTRIBUTIONS
FG-L, AA, and MM designed the experiment and interpreted
the results. AA performed the surgery. AA and DB processed
the tissue. AA and FG-L analyzed the data. AA, DB, and FG-L
wrote the manuscript. FG-L revised and expanded it throughout
the peer-review process. All authors contributed to manuscript
revision, read and approved the submitted version.
FUNDING
This study was supported by grants from the Oskar
Fischer Project Fund to FG-L and the Darrell K Royal
Research Fund to MM.
ACKNOWLEDGMENTS
The authors thank Justin Williams and Bryan Barksdale for their
assistance with the animal work. Some of this data was previously
included as part of A. Auchter’s dissertation at the University of
Texas at Austin (Auchter, 2015).
REFERENCES
Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H.,
et al. (2008). Methylene blue delays cellular senescence and enhances key
mitochondrial biochemical pathways. FASEB J. 22, 703–712. doi: 10.1096/fj.07-
9610com
Auchter, A., Shumake, J., Gonzalez-Lima, F., and Monfils, M. H. (2017). Preventing
the return of fear using reconsolidation updating and methylene blue is
differentially dependent on extinction learning. Sci. Rep. 7:46071. doi: 10.1038/
srep46071
Auchter, A., Williams, J., Barksdale, B., Monfils, M. H., and Gonzalez-Lima, F.
(2014). Therapeutic benefits of methylene blue on cognitive impairment during
chronic cerebral hypoperfusion. J. Alzheimers Dis. 42, S525–S535. doi: 10.3233/
JAD-141527
Auchter, A. M. (2015). Targeting Mitochondria via Methylene Blue: Implications in
Memory Enhancement and Neuroprotection. Ph.D. dissertation, University of
Texas, Austin TX.
Bhurtel, S., Katila, N., Neupane, S., Srivastav, S., Park, P. H., and Choi, D. Y.
(2018). Methylene blue protects dopaminergic neurons against MPTP-induced
neurotoxicity by upregulating brain-derived neurotrophic factor. Ann. N. Y.
Acad. Sci. 1431, 58–71. doi: 10.1111/nyas.13870
Biju, K. C., Evans, R. C., Shrestha, K., Carlisle, D. C. B., Gelfond, J., and Clark, R. A.
(2018). Methylene blue ameliorates olfactory dysfunction and motor deficits in
a chronic MPTP/probenecid mouse model of Parkinson’s disease. Neuroscience
380, 111–122. doi: 10.1016/j.neuroscience.2018.04.008
Br¯
ıede, J., and Duburs, G. (2007). Protective effect of cerebrocrast on rat brain
ischaemia induced by occlusion of both common carotid arteries. Cell Biochem.
Funct. 25, 203–210. doi: 10.1002/cbf.1318
Bruchey, A. K., and Gonzalez-Lima, F. (2008). Behavioral, physiological and
biochemical hormetic responses to the autoxidizable dye methylene blue. Am.
J. Pharmacol. Toxicol. 3, 72–79. doi: 10.3844/ajptsp.2008.72.79
Cada, A., de la Torre, J. C., and Gonzalez-Lima, F. (2000). Chronic cerebrovascular
ischemia in aged rats: effects on brain metabolic capacity and behavior.
Neurobiol. Aging 21, 225–233. doi: 10.1016/s0197-4580(00)00116-0
Frontiers in Cellular Neuroscience | www.frontiersin.org 15 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 16
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
Callaway, N. L., Riha, P. D., Bruchey, A. K., Munshi, Z., and Gonzalez-Lima,
F. (2004). Methylene blue improves brain oxidative metabolism and memory
retention in rats. Pharmacol. Biochem. Behav. 77, 175–181. doi: 10.1016/j.pbb.
2003.10.007
Callaway, N. L., Riha, P. D., Wrubel, K. M., McCollum, D., and Gonzalez-Lima,
F. (2002). Methylene blue restores spatial memory retention impaired by an
inhibitor of cytochrome oxidase in rats. Neurosci. Lett. 332, 83–86. doi: 10.1016/
s0304-3940(02)00827-3
Choy, M., Ganesan, V., Thomas, D. L., Thornton, J. S., Proctor, E., King,
M. D., et al. (2006). The chronic vascular and haemodynamic response
after permanent bilateral common carotid occlusion in newborn and adult
rats. J. Cereb. Blood Flow Metab. 26, 1066–1075. doi: 10.1038/sj.jcbfm.960
0259
Cohen, J. (1988). Statistical Power Analysis for the Behavioural Sciences, 2nd Edn.
New York, NY: Lawrence Erlbaum Associates.
Conejo, N. M., Gonzalez-Pardo, H., Gonzalez-Lima, F., and Arias, J. L. (2010).
Spatial learning of the water maze: progression of brain circuits mapped with
cytochrome oxidase histochemistry. Neurobiol. Learn. Mem. 93, 362–371. doi:
10.1016/j.nlm.2009.12.002
de la Torre, J. C. (1999). Critical threshold cerebral hypoperfusion causes
Alzheimer’s disease. Acta Neuropathol. 98, 1–8. doi: 10.1007/s00401005
1044
de la Torre, J. C. (2000). Impaired cerebrovascular perfusion. Summary
of evidence in support of its causality in Alzheimer’s disease. Ann.
N. Y. Acad. Sci. 924, 136–152. doi: 10.1111/j.1749-6632.2000.tb
05572.x
de la Torre, J. C. (2012). Cardiovascular risk factors promote brain hypoperfusion
leading to cognitive decline and dementia. Cardiovasc. Psychiatry Neurol.
2012:367516. doi: 10.1155/2012/367516
de la Torre, J. C. (2016). Alzheimer’s Turning Point: A Vascular Approach to Clinical
Prevention. Cham: Springer.
de la Torre, J. C. (2018). The vascular hypothesis of Alzheimer’s disease: a key to
preclinical prediction of dementia using neuroimaging. J. Alzheimers Dis. 63,
35–52. doi: 10.3233/JAD-180004
de la Torre, J. C., Cada, A., Nelson, N., Davis, G., Sutherland, R. J., and Gonzalez-
Lima, F. (1997). Reduced cytochrome oxidase and memory dysfunction after
chronic brain ischemia in aged rats. Neurosci. Lett. 223, 165–168. doi: 10.1016/
s0304-3940(97)13421-8
de la Torre, J. C., and Fortin, T. (1991). Partial or global rat brain ischemia:
the SCOT model. Brain Res. Bull. 26, 365–372. doi: 10.1016/0361-9230(91)90
008-8
de la Torre, J. C., Fortin, T., Park, G. A., Butler, K. S., Kozlowski, P., Pappas,
B. A., et al. (1992). Chronic cerebrovascular insufficiency induces dementia-
like deficits in aged rats. Brain Res. 582, 186–195. doi: 10.1016/0006-8993(92)
90132-s
Dhar, S. S., and Wong-Riley, M. T. T. (2009). Coupling of energy metabolism and
synaptic transmission at the transcriptional level: role of nuclear respiratory
factor 1 in regulating both cytochrome c oxidase and NMDA glutamate receptor
subunit genes. J. Neurosci. 29, 483–492. doi: 10.1523/JNEUROSCI.3704-08.
2009
Driscoll, I., Howard, S., Prusky, G., Rudy, J., and Sutherland, R. (2005). Seahorse
wins all races: hippocampus participates in both linear and non-linear visual
discrimination learning. Behav. Brain Res. 164, 29–35. doi: 10.1016/j.bbr.2005.
05.006
Echevarria, D. J., Caramillo, E. M., and Gonzalez-Lima, F. (2016). Methylene blue
facilitates memory retention in zebrafish in a dose-dependent manner. Zebrafish
13, 489–494. doi: 10.1089/zeb.2016.1282
Erecinska, M., and Silver, I. A. (1989). ATP and brain function. J. Cereb. Blood Flow
Metab. 9, 2–19. doi: 10.1038/jcbfm.1989.2
Farkas, E., Institoris, A., Domoki, F., Mihaly, A., Luiten, P. G., and
Bari, F. (2004). Diazoxide and dimethyl sulphoxide prevent cerebral
hypoperfusion-related learning dysfunction and brain damage after carotid
artery occlusion. Brain Res. 1008, 252–258. doi: 10.1016/j.brainres.2004.
02.037
Farkas, E., Institóris, Á, Domoki, F., Mihály, A., and Bari, F. (2006). The effect of
pre- and post-treatment with diazoxide on the early phase of chronic cerebral
hypoperfusion in the rat. Brain Res. 1087, 168–174. doi: 10.1016/j.brainres.2006.
02.134
Farkas, E., Luiten, P., and Bari, F. (2007). Permanent, bilateral common carotid
artery occlusion in the rat: a model for chronic cerebral hypoperfusion-
related neurodegenerative diseases. Brain Res. Rev. 54, 162–180. doi: 10.1016/
j.brainresrev.2007.01.003
Farkas, E., and Luiten, P. G. M. (2001). Cerebral microvascular pathology in aging
and Alzheimer’s disease. Prog. Neurobiol. 64, 575–611. doi: 10.1016/s0301-
0082(00)00068-x
Fidalgo, C., Conejo, N. M., González-Pardo, H., and Arias, J. L. (2014). Dynamic
functional brain networks involved in simple visual discrimination learning.
Neurobiol. Learn. Mem. 114, 165–170. doi: 10.1016/j.nlm.2014.06.001
Friston, K. J. (1994). Functional and effective connectivity in neuroimaging: A
synthesis. Human Brain Mapping 2, 56–78. doi: 10.1002/hbm.460020107
Gonzalez-Lima, F., and Bruchey, A. K. (2004). Extinction memory improvement
by the metabolic enhancer methylene blue. Learn. Mem. 11, 633–640. doi:
10.1101/lm.82404
Gonzalez-Lima, F., and Auchter, A., (2015). Protection against neurodegeneration
with low-dose methylene blue and near-infrared light. Front. Cell Neurosci.
9:179. doi: 10.3389/fncel.2015.00179
Gonzalez-Lima, F., and Cada, A. (1994). Cytochrome oxidase activity in the
auditory system of the mouse: a qualitative and quantitative histochemical
study. Neuroscience 63, 559–578. doi: 10.1016/0306-4522(94)90550- 9
Gonzalez-Lima, F. (ed.) (1998). Cytochrome Oxidase in Neuronal Metabolism and
Alzheimer’s Disease. New York, NY: Plenum Press.
Gonzalez-Lima, F., and 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. doi: 10.1016/0006-8993(94)90836-2
Gonzalez-Lima, F., and McIntosh, A. R. (1994). Neural network interactions
related to auditory learning analyzed with structural equation modeling. Hum.
Brain Mapp. 2, 23–44. doi: 10.1002/hbm.460020105
Gonzalez-Lima, F., and McIntosh, A. R. (1995). Analysis of neural network
interactions related to associative learning using structural equation modeling.
Math. Comput. Simul. 40, 115–140. doi: 10.1016/0378-4754(95)00022-x
Haley, A. P., Forman, D. E., Poppas, A., Hoth, K. F., Gunstad, J., Jefferson,
A. L., et al. (2007). Carotid artery intima-media thickness and cognition in
cardiovascular disease. Int. J. Cardiol. 121, 148–154. doi: 10.1016/j.ijcard.2006.
10.032
Horwitz, B., Soncrant, T. T., and Haxby, J. V. (1992). “Covariance analysis of
functional interactions in the brain using metabolic and blood flow data,” in
Advances in Metabolic Mapping Techniques for Brain Imaging of Behavioral and
Learning Functions, Vol. 68, eds F. Gonzalez-Lima, T. H. Finkenstaedt, and H.
Scheich (Dordrecht: Kluwer Academic Publishers), 189–217. doi: 10.1007/978-
94-011-2712-7_7
Huang, L., Lu, J., Cerqueira, B., Liu, Y., Jiang, Z., and Duong, T. Q. (2018).
Chronic oral methylene blue treatment in a rat model of focal cerebral
ischemia/reperfusion. Brain Res. 1678, 322–329. doi: 10.1016/j.brainres.2017.
10.033
Huang, S., Du, F., Shih, Y. Y., Shen, Q., Gonzalez-Lima, F., and Duong, T. Q. (2013).
Methylene blue potentiates stimulus-evoked fMRI responses and cerebral
oxygen consumption during normoxia and hypoxia. Neuroimage 72, 237–242.
doi: 10.1016/j.neuroimage.2013.01.027
Lavinsky, D., Sarmento Arterni, N., Achaval, M., and Netto, C. A. (2006).
Chronic bilateral common carotid artery occlusion: a model for ocular ischemic
syndrome in the rat. Graefes Arch. Clin. Exp. Ophthalmol. 244, 199–204. doi:
10.1007/s00417-005-0006-7
Lee, R. B., and Urban, J. P. (2002). Functional replacement of oxygen by other
oxidants in articular cartilage. Arthritis. Rheum. 46, 3190–3200. doi: 10.1002/
art.10686
Liu, H. X., Zhang, J. J., Zheng, P., and Zhang, Y. (2005). Altered expression of MAP-
2, GAP-43, and synaptophysin in the hippocampus of rats with chronic cerebral
hypoperfusion correlates with cognitive impairment. Brain Res. Mol. Brain Res.
139, 169–177. doi: 10.1016/j.molbrainres.2005.05.014
Liu, J., Jin, D. Z., Xiao, L., and Zhu, X. Z. (2006). Paeoniflorin attenuates chronic
cerebral hypoperfusion-induced learning dysfunction and brain damage in rats.
Brain Res. 1089, 162–170. doi: 10.1016/j.brainres.2006.02.115
Martinez, J. L. Jr., Jensen, R. A., Vasquez, B. J., McGuinness, T., and McGaugh,
J. L. (1978). Methylene blue alters retention of inhibitory avoidance responses.
Psychobiology 6, 387–390. doi: 10.3758/bf03326744
Frontiers in Cellular Neuroscience | www.frontiersin.org 16 May 2020 | Volume 14 | Article 130
fncel-14-00130 May 18, 2020 Time: 14:3 # 17
Auchter et al. Methylene Blue Treats Cerebral Hypoperfusion
McIntosh, A. R., and Gonzalez-Lima, F. (1994). Structural equation modeling and
its application to network analysis in functional brain imaging. Hum. Brain
Mapp. 2, 2–22. doi: 10.1016/j.neuroimage.2009.05.078
Miclescu, A., Basu, S., and 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. doi: 10.1097/01.
CCM.0000242517.23324.27
Miclescu, A., Sharma, H. S., Martijn, C., and Wiklund, L. (2010). Methylene
blue protects the cortical blood–brain barrier against ischemia/reperfusion-
induced disruptions. Crit. Care Med. 38, 2199–2206. doi: 10.1097/CCM.
0b013e3181f26b0c
Nair,B., and Wong-Riley, M. T. (2016). Trans criptional regulation of brain-derived
neurotrophic factor coding exon IX: role of nuclear respiratory factor 2. J. Biol.
Chem. 291, 22583–22593. doi: 10.1074/jbc.M116.742304
Nelson, C., and Silverstein, F. S. (1994). Acute disruption of cytochrome oxidase
activity in brain in a perinatal rat stroke model. Pediatr. Res. 36, 12–19. doi:
10.1203/00006450-199407001-00003
Ohtaki, H., Fujimoto, T., Sato, T., Kishimoto, K., Fujimoto, M., Moriya, M., et al.
(2006). Progressive expression of vascular endothelial growth factor (VEGF)
and angiogenesis after chronic ischemic hypoperfusion in rat. Acta Neurochir.
Suppl. 96, 283–287.
Packard, M. G., Cahill, L., and McGaugh, J. L. (1994). Amygdala modulation of
hippocampal-dependent and caudate nucleus-dependent memory processes.
Proc. Natl. Acad. Sci. U.S.A. 91, 8477–8481.
Paxinos, G., and Watson, C. (1996). The Rat Brain in Stereotaxic Coordinates, 3rd
Edn. San Diego, CA: Academic Press.
Plaschke, K. (2005). Aspects of ageing in chronic cerebral oligaemia. Mechanisms
of degeneration and compensation in rat models. J. Neural Transm. 112,
393–413. doi: 10.1007/s00702-004- 0191-2
Prusky, G. T., West, P. W., and Douglas, R. M. (2000). Behavioral assessment of
visual acuity in mice and rats. Vision Res. 40, 2201–2209.
Riha, P. D., Bruchey, A. K., Echevarria, D. J., and Gonzalez-Lima, F. (2005).
Memory facilitation by methylene blue: dose-dependent effect on behavior and
brain oxygen consumption. Eur. J. Pharmacol. 511, 151–158. doi: 10.1016/j.
ejphar.2005.02.001
Riha, P. D., Rojas, J. C., and Gonzalez-Lima, F. (2011). Beneficial network effects of
methylene blue in an amnestic model. Neuroimage 54, 2623–2634. doi: 10.1016/
j.neuroimage.2010.11.023
Rodriguez, P., Singh, A. P., Malloy, K. E., Zhou, W., Barrett, D. W., Franklin, C. G.,
et al. (2017). Methylene blue modulates functional connectivity in the human
brain. Brain Imaging Behav. 11, 640–648. doi: 10.1007/s11682-016-9541-6
Rodriguez, P., Zhou, W., Barrett, D. W., Altmeyer, W., Gutierrez, J. E., Li, J.,
et al. (2016). Multimodal randomized functional MR imaging of the effects
of methylene blue in the human brain. Radiology 281, 516–526. doi: 10.1148/
radiol.2016152893
Rojas, J. C., Bruchey, A. K., and Gonzalez-Lima, F. (2012). Neurometabolic
mechanisms for memory enhancement and neuroprotection of methylene blue.
Prog. Neurobiol. 96, 32–45. doi: 10.1016/j.pneurobio.2011.10.007
Rojas, J. C., John, J. M., Lee, J., and Gonzalez-Lima, F. (2009a). Methylene blue
provides behavioral and metabolic neuroprotection against optic neuropathy.
Neurotox. Res. 15, 260–273. doi: 10.1007/s12640-009-9027-z
Rojas, J. C., Simola, N., Kermath, B. A., Kane, J. R., Schallert, T., and Gonzalez-
Lima, F. (2009b). Striatal neuroprotection with methylene blue. Neuroscience
163, 877–889. doi: 10.1016/j.neuroscience.2009.07.012
Sakata, J. T., Coomber, P., Gonzalez-Lima, F., and Crews, D. (2000). Functional
connectivity among limbic brain areas: differential effects of incubation
temperature and gonadal sex in the leopard gecko, Eublepharis macularius.
Brain Behav. Evol. 55, 139–151. doi: 10.1159/000006648
Salaris, S. C., Babbs, C. F., and Voorhees, W. D. (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.
Sarti, C., Pantoni, L., Bartolini, L., and Inzitari, D. (2002). Persistent impairment of
gait performances and working memory after bilateral common carotid artery
occlusion in the adult Wistar rat. Behav. Brain Res. 136, 13–20.
Schmidt-Kastner, R., Truettner, J., Lin, B., Zhao, W., Saul, I., Busto, R., et al.
(2001). Transient changes of brain-derived neurotrophic factor (BDNF) mRNA
expression in hippocampus during moderate ischemia induced by chronic
bilateral common carotid artery occlusion in the rat. Brain Res. Mol. Brain Res.
92, 157–166.
Shang, Y., Cheng, J., Oi, J., and Miao, H. (2005). Scutellaria flavonoid reduced
memory dysfunction and neuronal injury caused by permanent global ischemia
in rats. Pharmacol. Biochem. Behav. 82, 67–73. doi: 10.1016/j.pbb.2005.06.018
Shen, Q., Du, F., Huang, S., Rodriguez, P., Watts, L. T., and Duong, T. Q. (2013).
Neuroprotective efficacy of methylene blue in ischemic stroke: an MRI study.
PLoS One 8:e79833. doi: 10.1371/journal.pone.0079833
Smith, E. S., Clark, M. E., Hardy, G. A., Kraan, D. J., Biondo, E., Gonzalez-Lima,
F., et al. (2017). Daily consumption of methylene blue reduces attentional
deficits and dopamine reduction in a 6-OHDA model of Parkinson’s disease.
Neuroscience 359, 8–16. doi: 10.1016/j.neuroscience.2017.07.001
Tachibana, H., Meyer, J. S., Okayasu, H., Shaw, T. G., Kandula, P., and Rogers,
R. L. (1984). Xenon contrast CT–CBF scanning of the brain differentiates
normal age-related changes from multi-infarct dementia and senile dementia
of Alzheimer type. J. Gerontol. 39, 415–423.
Telch, M. J., Bruchey, A. K., Rosenfield, D., Cobb, A. R., Smits, J., Pahl, S.,
et al. (2014). Effects of post-session administration of methylene blue on
fear extinction and contextual memory in adults with claustrophobia. Am. J.
Psychiatry 171, 1091–1098. doi: 10.1176/appi.ajp.2014.13101407
Tian, W. F., Zeng, S., Sheng, Q., Chen, J. L., Weng, P., Zhang, X. T., et al. (2018).
Methylene blue protects the isolated rat lungs from ischemia-reperfusion injury
by attenuating mitochondrial oxidative damage. Lung 196, 73–82. doi: 10.1007/
s00408-017-0072-8
Tucker, D., Lu, Y., and Zhang, Q. (2018). From mitochondrial function to
neuroprotection-an emerging role for methylene blue. Mol. Neurobiol. 55,
5137–5153. doi: 10.1007/s12035-017- 0712-2
Vélez-Hernández, M. E., Padilla, E., Gonzalez-Lima, F., and Jiménez-Rivera, C. A.
(2014). Cocaine reduces cytochrome oxidase activity in the prefrontal cortex
and modifies its functional connectivity with brainstem nuclei. Brain Res. 1542,
56–69. doi: 10.1016/j.brainres.2013.10.017
Villarreal, V. S., Gonzalez-Lima, F., Berndt, J., and Barea-Rodriguez, E. J. (2002).
Water maze training in aged rats: effects on brain metabolic capacity and
behavior. Brain Res. 939, 43–51.
Wong-Riley, M. T. (1989). Cytochrome oxidase: an endogenous metabolic marker
for neuronal activity. Trends Neursci. 12, 94–101.
Wong-Riley, M. T. (2012). Bigenomic regulation of cytochrome c oxidase
in neurons and the tight coupling between neuronal activity and energy
metabolism. Adv. Exp. Med. Biol. 748, 283–304. doi: 10.1007/978-1- 4614-3573-
0_12
Wong-Riley, M. T., and Welt, C. (1980). Histochemical changes in cytochrome
oxidase of cortical barrels after vibrissal removal in neonatal and adult mice.
Proc. Natl. Acad. Sci. U.S.A. 77, 2333–2337.
Wrubel, K. M., Riha, P. D., Maldonado, M. A., McCollum, D., and Gonzalez-
Lima, F. (2007). The brain metabolic enhancer methylene blue improves
discrimination learning in rats. Pharmacol. Biochem. Behav. 86, 712–717. doi:
10.1016/j.pbb.2007.02.018
Zhang, X., Rojas, J. C., and Gonzalez-Lima, F. (2006). Methylene blue prevents
neurodegeneration caused by rotenone in the retina. Neurotox. Res. 9, 47–57.
Zoellner, L. A., Telch, M., Foa, E. B., Farach, F. J., McLean, C. P., Gallop,
R., et al. (2017). Enhancing extinction learning in posttraumatic stress
disorder with brief daily imaginal exposure and methylene blue: a randomized
controlled trial. J. Clin. Psychiatry 78, e782–e789. doi: 10.4088/JCP.16m
10936
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Auchter, Barrett, Monfils and Gonzalez-Lima. This is an open-
access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.
Frontiers in Cellular Neuroscience | www.frontiersin.org 17 May 2020 | Volume 14 | Article 130
... Then we used the averaged regional data from each functional neuroanatomical system to calculate standard Pearson's bivariate correlations (r-values from 1 to 0) to create correlation matrices among the systems for each of the groups. This correlative analysis using CCO activity data has been established previously as a way to estimate the modification of network functional connectivity in the rat brain (Riha et al., 2011;Vélez-Hernández et al., 2014;Auchter et al., 2020). All analyses were performed using Excel and the Statistical Package for the Social Sciences (SPSS Inc., IBM, version 221.0, Chicago, IL, United States). ...
Article
Full-text available
In cellular bioenergetics, cytochrome c oxidase (CCO) is the enzyme responsible for oxygen consumption in the mitochondrial electron transport chain, which drives oxidative phosphorylation for adenosine triphosphate (ATP) production. CCO is also the major intracellular acceptor of photons in the light wavelengths used for photobiomodulation (PBM). Brain function is critically dependent on oxygen consumption by CCO for ATP production. Therefore, our objectives were (1) to conduct the first detailed brain mapping study of the effects of PBM on regional CCO activity, and (2) to compare the chronic effects of PBM on young and aged brains. Specifically, we used quantitative CCO histochemistry to map the differences in CCO activity of brain regions in healthy young (4 months old) and aged (20 months old) rats from control groups with sham stimulation and from treated groups with 58 consecutive days of transcranial laser PBM (810 nm wavelength and 100 mW power). We found that aging predominantly decreased regional brain CCO activity and systems-level functional connectivity, while the chronic laser stimulation predominantly reversed these age-related effects. We concluded that chronic PBM modified the effects of aging by causing the CCO activity on brain regions in laser-treated aged rats to reach levels similar to those found in young rats. Given the crucial role of CCO in bioenergetics, PBM may be used to augment brain and behavioral functions of older individuals by improving oxidative energy metabolism.
... Furthermore, the sociability and social novelty were increased in the social interaction test after pretreatment with both LLLT and MB. As other studies have reported, the results of the behavioral tests such as the visual water maze task [46], Morris water maze test [47], Barnes Maze Task [28], and What-Where-Which task [48] showed the beneficial effects of LLLT or MB on learning and memory. ...
Article
Full-text available
Low-level laser therapy (LLLT) and methylene blue (MB) were proved to have neuroprotective effects. In this study, we evaluated the preventive effects of LLLT and MB alone and in combination to examine their efficacy against sleep deprivation (SD)–induced cognitive impairment. Sixty Balb/c male mice were randomly divided into five groups as follows: wide platform (WP), SD, LLLT, MB, LMB (treatment with both LLLT and MB). Daily MB (0.5 mg/kg) was injected for ten consecutive days. An 810-nm, 10-Hz pulsed laser was used in LLLT every other day. We used the T-maze test, social interaction test (SIT), and shuttle box to assess learning and memory and PSD-95, GAP-43, and synaptophysin (SYN) markers to examine synaptic proteins levels in the hippocampus. Our results showed that SD decreased alternation rate in the T-maze test, sociability and social novelty in SIT, and memory index in the shuttle box. Single treatments were not able to reverse these in most of the behavioral parameters. However, behavioral tests showed a significant difference between combined therapy and the SD group. The levels of synaptic plasticity markers were also significantly reduced after SD. There was a significant difference between the MB group and SD animals in GAP-43 and SYN biomarkers. Combination treatment with LLLT and MB also increased GAP-43, PSD-95, and SYN compared to the SD group. We found that the combined use of LLLT and MB pretreatment is more effective in protecting SD-induced cognitive impairment, which may be imparted via modulation of synaptic proteins.
... These effects were accompanied by long-lasting mitochondrial respiratory function [33]. Another study showed that MB could prevent memory impairment in rats with chronic cerebral hypoperfusion [76]. With functional imaging in the human brain, it was shown that MB could modulate task-related and resting-state neural networks [77]. ...
Article
Full-text available
Methylene blue (MB), as the first fully man-made medicine, has a wide range of clinical applications. Apart from its well-known applications in surgical staining, malaria, and methemoglobinemia, the anti-oxidative properties of MB recently brought new attention to this century-old drug. Mitochondrial dysfunction has been observed in systematic aging that affects many different tissues, including the brain and skin. This leads to increaseding oxidative stress and results in downstream phenotypes under age-related conditions. MB can bypass Complex I/III activity in mitochondria and diminish oxidative stress to some degree. This review summarizes the recent studies on the applications of MB in treating age-related conditions, including neurodegeneration, memory loss, skin aging, and a premature aging disease, progeria.
... This dye can sup press aggregation of tau protein, acts as an antioxidant and cofactor of the mitochondria targeted catalase, thereby minimizing the excessive generation of ROS [86]. It was first shown in the chronic cerebral hypoperfusion model in rats, that methylene blue prevented neurode generation and memory impairment by maintaining cytochrome oxidase activity [92]. ...
Article
Full-text available
Alzheimer’s disease is the most common age-related neurodegenerative disease. Understanding of its etiology and pathogenesis is constantly expanding. Thus, the increasing attention of researchers is directed to the study of the role of mitochondrial disorders. In addition, in recent years, the concept of Alzheimer’s disease as a stress-induced disease has begun to form more and more actively. The stress-induced damage to the neuronal system can trigger a vicious circle of pathological processes, among which mitochondrial dysfunctions have a significant place, since mitochondria represent a substantial component in the anti-stress activity of the cell. The study of mitochondrial disorders in Alzheimer’s disease is relevant for at least two reasons: first, as important pathogenetic component in this disease; second, due to vital role of mitochondria in formation of the body resistance to various conditions, including stressful ones, throughout the life. This literature review analyzes the results of a number of recent studies assessing potential significance of the mitochondrial disorders in Alzheimer’s disease. The probable mechanisms of mitochondrial disorders associated with the development of this disease are considered: bioenergetic dysfunctions, changes in mitochondrial DNA (including assessment of the significance of its haplogroup features), disorders in the dynamics of these organelles, oxidative damage to calcium channels, damage to MAM complexes (membranes associated with mitochondria; mitochondria-associated membranes), disruptions of the mitochondrial quality control system, mitochondrial permeability, etc. The issues of the “primary” or “secondary” mitochondrial damage in Alzheimer’s disease are discussed. Potentials for the development of new methods for diagnosis and therapy of mitochondrial disorders in Alzheimer’s disease are considered.
Article
Gender is considered as a pivotal determinant of mental health. Indeed, several psychiatric disorders such as anxiety and depression are more common and persistent in women than in men. In the past two decades, impaired brain energy metabolism has been highlighted as a risk factor for the development of these psychiatric disorders. However, comprehensive behavioural and neurobiological studies in brain regions relevant to anxiety and depression symptomatology are scarce. In the present study, we summarize findings describing cannabidiol effects on anxiety and depression in maternally separated female mice as a well-established rodent model of early-life stress associated with many mental disorders. Our results indicate that cannabidiol could prevent anxiolytic- and depressive-related behaviour in early-life stressed female mice. Additionally, maternal separation with early weaning caused long-term changes in brain oxidative metabolism in both nucleus accumbens and amygdalar complex measured by cytochrome c oxidase quantitative histochemistry. However, cannabidiol treatment could not revert brain oxidative metabolism impairment. Moreover, we identified hyperphosphorylation of mTOR and ERK 1/2 proteins in the amygdala but not in the striatum, that could also reflect altered brain intracellular signalling related with to bioenergetic impairment. Altogether, our study supports the hypothesis that MSEW induces profound long-lasting molecular changes in mTOR signalling and brain energy metabolism related to depressive-like and anxiety-like behaviours in female mice, which were partially ameliorated by CBD administration.
Article
Full-text available
Introduction: Impaired mitochondrial function is a key factor attributing to the lung ischemia reperfusion injury (LIRI). Methylene blue (MB) has been reported to attenuate brain and renal ischemia-reperfusion injury. We hypothesized that MB also could have a protective effect against LIRI by preventing mitochondrial oxidative damage. Methods: Isolated rat lungs were assigned to the following four groups (n = 6): a sham group: perfusion for 105 min without ischemia; I/R group: shutoff of perfusion and ventilation for 45 min followed by reperfusion for 60 min; and I/R + MB group and I/R + glutathione (GSH) group: 2 mg/kg MB or 4 μM glutathione were intraperitoneally administered for 2 h, and followed by 45 min of ischemia and 60 min of reperfusion. Results: MB lessened pulmonary dysfunction and severe histological injury induced by ischemia-reperfusion injury. MB reduced the production of reactive oxygen species and malondialdehyde and enhanced the activity of superoxide dismutase. MB also suppressed the opening of the mitochondrial permeability transition pore and partly preserved mitochondrial membrane potential. Moreover, MB inhibited the release of cytochrome c from the mitochondria into the cytosol and decreased apoptosis. Additionally, MB downregulated the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-18). Conclusion: MB protects the isolated rat lungs against ischemia-reperfusion injury by attenuating mitochondrial damage.
Article
Full-text available
Methylene blue (MB) is a well-established drug with a long history of use, owing to its diverse range of use and its minimal side effect profile. MB has been used classically for the treatment of malaria, methemoglobinemia, and carbon monoxide poisoning, as well as a histological dye. Its role in the mitochondria, however, has elicited much of its renewed interest in recent years. MB can reroute electrons in the mitochondrial electron transfer chain directly from NADH to cytochrome c, increasing the activity of complex IV and effectively promoting mitochondrial activity while mitigating oxidative stress. In addition to its beneficial effect on mitochondrial protection, MB is also known to have robust effects in mitigating neuroinflammation. Mitochondrial dysfunction has been identified as a seemingly unifying pathological phenomenon across a wide range of neurodegenerative disorders, which thus positions methylene blue as a promising therapeutic. In both in vitro and in vivo studies, MB has shown impressive efficacy in mitigating neurodegeneration and the accompanying behavioral phenotypes in animal models for such conditions as stroke, global cerebral ischemia, Alzheimer’s disease, Parkinson’s disease, and traumatic brain injury. This review summarizes recent work establishing MB as a promising candidate for neuroprotection, with particular emphasis on the contribution of mitochondrial function to neural health. Furthermore, this review will briefly examine the link between MB, neurogenesis, and improved cognition in respect to age-related cognitive decline.
Article
Full-text available
Recently, alternative drug therapies for Parkinson’s disease (PD) have been investigated as there are many shortcomings of traditional dopamine-based therapies including difficulties in treating cognitive and attentional dysfunction. A promising therapeutic avenue is to target mitochondrial dysfunction and oxidative stress in PD. One option might be the use of methylene blue (MB), an antioxidant and metabolic enhancer. MB has been shown to improve cognitive function in both intact rodents and rodent disease models. Therefore, we investigated whether MB might treat attentional deficits in a rat model of PD induced by 6-hydroxydopamine (6-OHDA). MB also has neuroprotective capabilities against neurotoxic insult, so we also assessed the ability of MB to provide neuroprotection in our PD model. The results show that MB could preserve some dopamine neurons in the substantia nigra par compacta when 6-OHDA was infused into the medial forebrain bundle. This neuroprotection did not yield a significant behavioral improvement when motor functions were measured. However, MB significantly improved attentional performance in the five-choice task designed to measure selective and sustained attention. In conclusion, MB might be useful in improving some attentional function and preserving dopaminergic cells in this model. Future work should continue to study and optimize the abilities of MB for the treatment of PD.
Article
Full-text available
Objective: The memory-enhancing drug methylene blue (MB) administered after extinction training improves fear extinction retention in rats and humans with claustrophobia. Robust findings from animal research, in combination with established safety and data showing MB-enhanced extinction in humans, provide a foundation to extend this work to extinction-based therapies for posttraumatic stress disorder (PTSD) such as prolonged exposure (PE). Methods: Patients with chronic PTSD (DSM-IV-TR; N = 42) were randomly assigned to imaginal exposure plus MB (IE + MB), imaginal exposure plus placebo (IE + PBO), or waitlist (WL/standard PE) from September 2011 to April 2013. Following 5 daily, 50-minute imaginal exposure sessions, 260 mg of MB or PBO was administered. Waitlist controls received PE following 1-month follow-up. Patients were assessed using the independent evaluator-rated PTSD Symptom Scale-Interview version (primary outcome), patient-rated PTSD, trauma-related psychopathology, and functioning through 3-month follow-up. Results: Both IE + MB and IE + PBO showed strong clinical gains that did not differ from standard PE at 3-month follow-up. MB-augmented exposure specifically enhanced independent evaluator-rated treatment response (number needed to treat = 7.5) and quality of life compared to placebo (effect size d = 0.58). Rate of change for IE + MB showed a delayed initial response followed by accelerated recovery, which differed from the linear pattern seen in IE + PBO. MB effects were facilitated by better working memory but not by changes in beliefs. Conclusions: The findings provide preliminary efficacy for a brief IE treatment for PTSD and point to the potential utility of MB for enhancing outcome. Brief interventions and better tailoring of MB augmentation strategies, adjusting for observed patterns, may have the potential to reduce dropout, accelerate change, and improve outcomes. Trial registration: ClinicalTrials.gov identifier: NCT01188694.
Article
Full-text available
Many factors account for how well individuals extinguish conditioned fears, such as genetic variability, learning capacity and conditions under which extinction training is administered. We predicted that memory-based interventions would be more effective to reduce the reinstatement of fear in subjects genetically predisposed to display more extinction learning. We tested this hypothesis in rats genetically selected for differences in fear extinction using two strategies: (1) attenuation of fear memory using post-retrieval extinction training, and (2) pharmacological enhancement of the extinction memory after extinction training by low-dose USP methylene blue (MB). Subjects selectively bred for divergent extinction phenotypes were fear conditioned to a tone stimulus and administered either standard extinction training or retrieval + extinction. Following extinction, subjects received injections of saline or MB. Both reconsolidation updating and MB administration showed beneficial effects in preventing fear reinstatement, but differed in the groups they targeted. Reconsolidation updating showed an overall effect in reducing fear reinstatement, whereas pharmacological memory enhancement using MB was an effective strategy, but only for individuals who were responsive to extinction.
Article
The relatively old, yet clinically used, drug methylene blue (MB) is known to possess neuroprotective properties by reducing aggregated proteins, augmenting the antioxidant response, and enhancing mitochondrial function and survival in various models of neurodegenerative diseases. In this study, we aimed to examine the effects of MB in Parkinson's disease (PD) in vivo and in vitro models by using 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)/1‐methyl‐4‐phenylpyridinium (MPP+) with a focus on possible effects on induction of neurotrophic factors. Our results indicate that pretreatment with MB significantly attenuated MPTP‐induced loss of dopaminergic neurons, glial cell activation, and depletion of dopamine. We also found that MB upregulated brain‐derived neurotrophic factor (BDNF) and activated its downstream signaling pathways, suggesting that BDNF might be a contributor to MB‐associated neuroprotection. Specific inhibition of the BDNF receptor or extracellular signal‐regulated kinase (Erk) reversed the MB‐mediated protection against MPP+ toxicity, thus implying a role for BDNF and the Erk pathway in the neuroprotective effects. Taken together, our data suggest that MB protects neurons from MPTP neurotoxicity via induction of BDNF. Further study to determine whether MB preserves dopaminergic neurons in the brains of PD patients is warranted. There has been no research, to our knowledge, on the effects of methylene blue (MB) on neurotrophic factors in a Parkinson's disease (PD) model. In the present study, we utilize a subchronic model of MPTP‐induced PD to evaluate the neuroprotective effects of MB, possibly involving neurotrophic factor induction.
Article
Mitochondrial dysfunction and oxidative stress are very prominent and early features in Parkinson's disease (PD) and in animal models of PD. Thus, antioxidant therapy for PD has been proposed, but in clinical trials such strategies have met with very limited success. Methylene blue (MB), a small-molecule synthetic heterocyclic organic compound that acts as a renewable electron cycler in the mitochondrial electron transport chain, manifesting robust antioxidant and cell energetics-enhancing properties, has recently been shown to have significant beneficial effects in reducing nigrostriatal dopaminergic loss and motor impairment in acute toxin models of PD. However, no studies have investigated the impact of this promising agent in chronic models or for olfactory dysfunction, an early non-motor feature of PD. To test the efficacy of low-dose MB for olfactory dysfunction, motor symptoms, and dopaminergic neurodegeneration, mice were injected with ten subcutaneous doses of 25 mg/kg MPTP, plus 250 mg/kg intraperitoneal probenecid or saline/probenecid at 3.5-day intervals. Following the onset of olfactory dysfunction, MPTP/probenecid (MPTP/p) and saline/probenecid mice were provided drinking water with or without 1 mg/kg/day MB. Oral delivery of low-dose MB significantly ameliorated MPTP/p-induced deficits in motor coordination, as well as degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH-positive terminals in the striatum. Importantly, olfactory dysfunction was ameliorated by MB treatment, whereas this benefit is not observed with currently available anti-Parkinsonian medications. These results indicate that low-dose MB is a promising neuroprotective intervention for both motor and non-motor features of PD.
Article
The vascular hypothesis of Alzheimer's disease (VHAD) was proposed 24 years ago from observations made in our laboratory using aging rats subjected to chronic brain hypoperfusion. In recent years, VHAD has become a mother-lode to numerous neuroimaging studies targeting cerebral hemodynamic changes, particularly brain hypoperfusion in elderly patients at risk of developing Alzheimer's disease (AD). There is a growing consensus among neuroradiologists that brain hypoperfusion is likely involved in the pathogenesis of AD and that disturbed cerebral blood flow (CBF) can serve as a key biomarker for predicting conversion of mild cognitive impairment to AD. The use of cerebral hypoperfusion as a preclinical predictor of AD is becoming decisive in stratifying low and high risk patients that may develop cognitive decline and for assessing the effectiveness of therapeutic interventions. There is currently an international research drive from neuroimaging groups to seek new perspectives that can broaden our understanding of AD and improve lifestyle. Diverse neuroimaging methods are currently being used to monitor normal and dyscognitive brain activity. Some techniques are very powerful and can detect, diagnose, quantify, prognose, and predict cognitive decline before AD onset, even from a healthy cognitive state. Multimodal imaging offers new insights in the treatment and prevention of cognitive decline during advanced aging and better understanding of the functional and structural organization of the human brain. This review discusses the impact the VHAD and CBF are having on the neuroimaging technology that can usher practical strategies to help prevent AD.
Book
This book is based on an international symposium titled "Cytochrome oxidase in energy metabolism and Alzheimer's disease," held as a satellite to the 27th meeting of the Society for Neuroscience, New Orleans, 1997. The symposium was dedicated in honor of Dr. Margaret T. T. Wong-Riley because, in our opinion, the cytochrome oxidase histo­ chemical method introduced by Dr. Wong-Riley in 1979 was the most significant break­ through to map energy metabolism in the entire brain since the 2-deoxyglucose method introduced by Dr. Louis Sokoloff and colleagues in 1977. Both of these metabolic map­ ping techniques have made monumental contributions to brain research by allowing an integral view of brain activity. They have also developed into various specialized tech­ niques, including applications to the human brain. One of these new applications, which is described in detail in this book, is the quantitative cytochrome oxidase cytochemical method used to study Alzheimer's disease. The objective of this book is to describe the role of cytochrome oxidase in neuronal metabolism and Alzheimer's disease. Whether genetic or environmental, the pathogenesis of Alzheimer's disease involves a cascade of multiple intracellular events, eventually re­ sulting in failure of oxidative energy metabolism. Could impairment of cytochrome oxi­ dase in energy metabolism initiate the degenerative process? Cytochrome oxidase function and dysfunction are discussed in relationship to neuronal energy metabolism, neurodegen­ eration, and Alzheimer's disease. The book is made up of 10 chapters, divided into three major parts.
Article
A single acute low-dose methylene blue (MB), an FDA-grandfathered drug, has been shown to ameliorate behavioral deficits and reduces MRI-defined infarct volume in experimental ischemic stroke when administered intravenously or intraperitoneally. The efficacy of chronic MB treatment in ischemic stroke remains unknown. In a randomized, double-blinded and vehicle-controlled design, we investigated the efficacy of chronic oral MB administration in ischemic stroke longitudinally up to 60 days post injury using MRI and behavioral tests, with end-point histology. The major findings were chronic oral MB treatment, compared to vehicle, i) improves functional behavioral outcomes starting on day 7 and up to 60 days, ii) reduces MRI-defined total lesion volumes from day 14 and up to 60 days where some initial abnormal MRI-defined core and perfusion-diffusion mismatch were salvaged, iii) reduces white-matter damage, iv) gray matter and white matter damages are consistent with Nissl stains and Black Gold stain histology. These findings provide further evidence that long-term oral administration of low-dose MB is safe and has positive therapeutic effects in chronic ischemic stroke.