biomedicines

Article
Methylene Blue Reduces Electroretinogram Distortion and
Ganglion Cell Death in a Rat Model of Glaucoma
Ronan Nakamura 1 , Nicolás S. Ciranna 1 , Juan C. Fernández 1 , Rafael Peláez 2 , Álvaro Pérez-Sala 2 ,
Miriam Bobadilla 2 , Juan J. López-Costa 1 , César F. Loidl 1 , Alfredo Martínez 3, *,† and Manuel Rey-Funes 1,†

1 Institute of Cell Biology and Neurosciences “Prof. E. De Robertis”, Faculty of Medicine, University of Buenos
Aires, Buenos Aires C1121ABG, Argentina; [email protected] (R.N.); [email protected] (N.S.C.);
[email protected] (J.C.F.); [email protected] (J.J.L.-C.); [email protected] (C.F.L.);
[email protected] (M.R.-F.)
2 Biomarkers and Molecular Signaling Group, Neurodegenerative Diseases Area, Center for Biomedical
Research of La Rioja (CIBIR), 26006 Logroño, Spain; [email protected] (R.P.);
[email protected] (Á.P.-S.); [email protected] (M.B.)
3 Angiogenesis Group, Oncology Area, Center for Biomedical Research of La Rioja (CIBIR),
26006 Logroño, Spain
* Correspondence: [email protected]; Tel.: +34-941-278775
† These authors contributed equally to this work.

Abstract: Glaucoma is the second leading cause of blindness worldwide and is, in most cases,
a consequence of elevated intraocular pressure (IOP), ultimately resulting in the death of retinal
ganglion cells (RGCs). Current treatments are mostly focused on normalizing IOP, but we propose
the additional use of neuroprotective agents, including methylene blue (MB), to block the loss of
RGCs. Wistar rats were subjected to episcleral vein cauterization (EVC) in the left eye while the right
eye was sham-operated. One week later, they were divided into two groups, which were injected
Citation: Nakamura, R.; Ciranna, N.S.;
Fernández, J.C.; Peláez, R.; Pérez-Sala,
with either 2.0 mg/kg MB or phosphate-buffered saline (PBS), twice a day, for 7 days. Fifteen days
Á.; Bobadilla, M.; López-Costa, J.J.; after surgery, rats were tested with scotopic electroretinography (ERG) or pattern electroretinography
Loidl, C.F.; Martínez, A.; Rey-Funes, (PERG). After sacrifice, the number of RGCs and the thickness of the inner retina (IR) were evaluated
M. Methylene Blue Reduces both in the peripheral and central areas of the retina. Scotopic ERG showed a marked reduction
Electroretinogram Distortion and (p < 0.0001) on the a- and b-wave amplitude and oscillatory potential (OP) complexity of the eyes
Ganglion Cell Death in a Rat Model of subjected to EVC. These parameters were significantly (p < 0.01) restored by the application of MB.
Glaucoma. Biomedicines 2024, 12, 1983. PERG indicated that EVC was responsible for a very significant decrease in N2 amplitude (p < 0.0001)
https://doi.org/10.3390/ and prolongation of N2 implicit time (p < 0.0001). Treatment with MB significantly restored N2
biomedicines12091983
amplitude (p < 0.0001). In parallel with the ERG results, morphological analysis showed a significant
Academic Editors: Monika Wujec, loss of RGCs (p < 0.0001) and IR thickness (p < 0.0001) in both the peripheral and central retinas
Anna Bogucka-Kocka, subjected to EVC, which was significantly prevented (p < 0.0001) by MB treatment. We have shown
Przemysław Kołodziej and that MB treatment can be effective in preventing physiological and morphological hallmarks of optic
Jacek Bogucki neuropathy in a model of ocular hypertension, which faithfully recapitulates human open-angle
Received: 9 August 2024 glaucoma. Due to its high safety profile, this drug could therefore represent a new pharmacologic
Revised: 21 August 2024 strategy to prevent vision loss in glaucoma patients.
Accepted: 23 August 2024
Published: 2 September 2024 Keywords: glaucoma; episcleral vein cauterization; methylene blue; rat model; scotopic
electroretinography; pattern electroretinography; morphological analysis; inner retina thickness

Copyright: © 2024 by the authors.

Licensee MDPI, Basel, Switzerland. 1. Introduction
This article is an open access article
Optic neuropathy-related ocular lesions are responsible for major lifelong disabilities
distributed under the terms and
conditions of the Creative Commons
worldwide. This visual impairment implies significant morbidity for patients and an
Attribution (CC BY) license (https://
increasing economic burden to the health system and society in general. Glaucoma is
creativecommons.org/licenses/by/ the second leading cause of blindness worldwide, following cataracts. In 2020, around
4.0/). 76 million people were affected by this pathology, and these numbers are expected to rise

Biomedicines 2024, 12, 1983. https://doi.org/10.3390/biomedicines12091983 https://www.mdpi.com/journal/biomedicines

Biomedicines 2024, 12, 1983 2 of 15

to 111 million by the year 2040 [1]. Therefore, developing new treatment strategies for
preventing vision losses becomes crucial.
Glaucoma constitutes a heterogeneous group of degenerative retinopathies affecting
the inner retina. The histopathologic hallmark of these pathologies is the loss of retinal
ganglion cells (RGCs) and concomitant thinning of the optic nerve fibre layer and optic
nerve cupping. Because of the disposition of axons at the emergence of the optic nerve, this
translates into progressive and irreversible loss of peripheral vision and eventually, in late
stages, loss of central visual field and visual acuity. Given the oligosymptomatic nature
of this disease, glaucoma is usually diagnosed at late stages when RGC loss is already
significant [2].
There are many types of glaucoma, which are mostly classified in function of the
pathophysiological mechanism of production and intraocular pressure (IOP) values, the
latter accounting for the main and most frequent cause of glaucomatous degeneration.
The most frequent form of glaucoma is primary open-angle glaucoma (POAG) which
is responsible for more than 80% of cases [3,4]. The remaining forms include primary
closed-angle glaucoma (PCAG), normotensive glaucoma and other causes of secondary
glaucoma [5].
There are multiple aetiologies of glaucoma, but the main hallmarks of glaucoma
are IOP elevation, vascular dysfunction and neurodegeneration, among others. Even
though multiple theories have been proposed, currently there is a significant lack of
advances related to the neurodegenerative process and the vascular component of glaucoma
development. Also, the connection between the pressure gradient installed in the anterior
segment and the development of optic neuropathy in the posterior segment is still a matter
of study. The existence of normotensive forms of glaucoma, as well as the progression
of glaucomatous changes after IOP has been normalized, supports the need to develop
new therapeutic strategies, beyond IOP control, aimed to prevent the neurodegenerative
process in the retina, which is more than just a mere sign of ocular hypertension [2].
Many experimental animal models have been established to study glaucoma patho-
physiology and treatment. One of the most used models, probably because it best re-
capitulates the most common human form of glaucoma (POAG), is the episcleral vein
cauterization (EVC) model, which was first proposed in 1995 [6]. This murine model has
allowed significant advances in glaucoma knowledge, has been reproduced by multiple
research groups [7–9], and has also been applied to other mammalian species [10].
The current paradigm of glaucoma treatment focuses exclusively in normalizing
elevated IOP values, either through pharmacological (beta blockers, carbon anhydrase
inhibitors, prostaglandins analogues, etc.) or, in some cases, surgical (iridotomy, laser
iridoplasty, laser trabeculoplasty, trabeculectomy, etc.) approaches [2]. Furthermore, in
the case of neovascular glaucoma, the use of anti-VEGF agents is recommended [11]. In
addition, a number of novel approaches for the medical management of glaucoma are being
investigated. These include the use of Rho kinase inhibitors, vasodilators, prostaglandin
analogues, biodegradable implants, and nanotechnology [12]. Paradoxically, there is no
approved treatment available to prevent or diminish the neurodegenerative retinal process,
which is the mechanism ultimately responsible for blindness.
The free radical nitric oxide (NO) has been recognized as an important intercellular
messenger in the eye and in the pathogenesis of glaucoma [13] but the effects of NO are
somewhat paradoxical, depending on its physiological source. On the one hand, low
levels of NO, such as those produced by the endothelial isoform of nitric oxide synthase
(eNOS), increase blood flow and, thus, are beneficial for eye physiology [14] and some
novel treatments based on providing low levels of NO through NO-donors are being
investigated [15]. On the other hand, the high levels of NO generated by the inducible
(iNOS), or occasionally the neuronal (nNOS), isoforms evoke neuronal cell death and
vision losses [16]. Animal models suggest that an increasing IOP results in higher levels
of NO in the eye that may induce RGC death [17,18]. In humans, evidence of increased
levels of the three isoforms of NOS as a consequence of glaucoma have been shown in
Biomedicines 2024, 12, 1983 3 of 15

the optic nerve head [19]. Furthermore, treatment of POAG patients and matched healthy
volunteers with a NOS inhibitor showed evidence of higher NO levels in the glaucoma
group [20]. In consequence, inhibition of excessive NO levels may constitute a new avenue
for glaucoma treatment and protection of the RGCs [21,22]. Most physiological actions of
NO are mediated by the formation of the second messenger cGMP, which is produced by
the enzyme guanylyl cyclase (GC) [23]. In previous studies, our group has demonstrated
that methylene blue (MB), a drug that is used in the clinic with a high safety profile [24], is
a guanylyl cyclase inhibitor, free radical scavenger [25], and inhibitor of both constitutive
and inducible isoforms of NOS [26,27], can protect the retina from RGC death [28,29]. The
proposed mechanism of action for this effect was the inhibition of NOS/GC/cGMP by MB,
followed by the reduction of NO levels, and blockade of nitrosative damage caused by
nitrogen reactive species [16].
The main objective of this work was to develop an efficacious treatment to prevent
glaucoma-related retinal lesions. For that purpose, the pharmacological effect and efficacy
of MB was evaluated in a rat EVC model of ocular hypertension, analysing its influence
through modifications in two hallmarks of optic neuropathy, e.g., electroretinogram and
morphological changes in the retina.

2. Materials and Methods

2.1. Ocular Hypertension Glaucoma Model Using Episcleral Vein Cauterization
Young (8-week-old) male Wistar rats (n = 50) with genetic quality and sanitary certifica-
tion from the animal facility of our institution were cared for in accordance with guidelines
published in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
All procedures were approved by the Ethical Committee of CICUAL (Comité Institucional
para el Uso y Cuidado de Animales de Laboratorio, Facultad de Medicina, Universidad de
Buenos Aires, Buenos Aires, Argentina. Resolution RESCD-2023-1408-E-UBA-DCT#FMED).
Animals were kept under standard laboratory conditions, with light/dark cycles of 12/12 h,
a constant temperature of 21.0 ± 2.0 ◦ C, and food and water provided ad libitum.
The EVC protocol was performed as described [6], with slight modifications. Briefly,
rats were anesthetized with an intraperitoneal injection of 40 mg/kg ketamine (Ketamina
50, Holliday-Scott S.A., Buenos Aires, Argentina) and 5 mg/kg xylazine (Xilacina 20 Rich-
mond, Laboratorios Richmond, Buenos Aires, Argentina). A small incision was performed
on the superior bulbar conjunctiva, and then another on the Tenon capsule, of the left eye.
Connective tissue was carefully dissected and episcleral veins were exposed (Figure 1). The
two episcleral veins adjacent to the superior rectus muscle were identified by pupillary
transillumination. They were immediately cauterized using a hand-held electrocautery
(Bovie Medical Corp., Cleanwater, FL, USA), taking caution to not produce excessive ther-
mic damage on the adjacent tissues. Surgical wound closure was performed by layers. The
contralateral eye was sham-operated: the episcleral veins were exposed but not cauterized.
After surgery, intraperitoneal Tramadol 5 mg/kg (John Martin S.R.L., Buenos Aires, Ar-
gentina) and topic ophthalmic eritromicin ointment (Eritromicina Elea, Elea, Buenos Aires,
Argentina) were applied as analgesic and antibiotic prophylaxis, respectively. Animals
were checked daily for signs of pain or discomfort.
All animals were subjected to EVC in the left eye, whereas the right eye was sham-
operated, as described [6] (Figure 1).
Biomedicines 2024, 12, x FOR PEER REVIEW 4 of 15
Biomedicines 2024, 12, 1983 4 of 15

Figure
Figure1.1.Schematic
Schematic drawing
drawing of the localization
localization of
ofthe
theepiscleral
episcleralveins
veinsononthe
the
eyeeye and
and effect
effect of the
of the
cauterization
cauterization procedure (upper row).
procedure (upper row).Small
Smallblack
blackarrows
arrowsindicate
indicatethe
the cauterized
cauterized veins.
veins. SR:SR: superior
superior
rectus
rectusmuscle, SO: superior
muscle, SO: superioroblique
obliquemuscle,
muscle,
MR:MR: medial
medial rectus
rectus muscle,
muscle, IR: inferior
IR: inferior rectusrectus
muscle,muscle,
IO:
IO: inferior
inferior oblique
oblique muscle,
muscle, LR: lateral
LR: lateral rectus muscle.
rectus muscle. Actual photographs
Actual photographs of the are
of the procedure procedure
shown inare
shown in the
the lower lower
row. Size row.
bar =Size bar = 5 mm.
5 mm.

Seven
Seven days post-surgery,animals
days post-surgery, animalswere
were randomly
randomly distributed
distributed intointo
two two groups.
groups. Half Half
of
ofthethe
rats started
rats intraperitoneal
started treatment
intraperitoneal with 2.0
treatment with mg/kg MB (Sigma,
2.0 mg/kg St. Louis,
MB (Sigma, St.MO, USA),
Louis, MO,
twice twice
USA), a day afordayseven days, while
for seven days, the other
while thehalf
otherreceived the same
half received thevolume of vehicle
same volume of(PBS)
vehicle
(Figure 2). One day after finishing the treatments, all animals were
(PBS) (Figure 2). One day after finishing the treatments, all animals were subjected subjected to electro-
to elec-
physiological evaluation
trophysiological withwith
evaluation one of
onetwoofdifferent paradigms:
two different scotopicscotopic
paradigms: electroretinography
electroretino-
(ERG, n = 10 per group) or pattern electroretinography (PERG, n =
graphy (ERG, n = 10 per group) or pattern electroretinography (PERG, n = 15 per group) 15 per group) (see
below). Finally, all animals were sacrificed and tissues from the ERG group
(see below). Finally, all animals were sacrificed and tissues from the ERG group were col- were collected
for histological studies (see below).
lected for histological studies (see below).
Biomedicines
Biomedicines 2024, 12, 1983
2024, 12, x FOR PEER REVIEW 5 of 15
5 of 15

Figure 2.
Figure Schematic drawing
2. Schematic drawing ofof the
the experimental
experimentalprocedure.
procedure.The
Theleft
lefteyes
eyeswere
weresubjected
subjectedtotoepiscleral
episcle-
veinvein
ral cauterization (EVC)
cauterization whilewhile
(EVC) the right eyes were
the right eyes sham-operated. Seven days
were sham-operated. Sevenlater,
days rats were
later, injected,
rats were
twice a day
injected, fora 7day
twice days,
for with either
7 days, withPBS or PBS
either 2.0 mg/kg MB. One
or 2.0 mg/kg MB.day
Oneafter finishing
day after the treatment,
finishing the treat-
animals
ment, were subjected
animals to ERG
were subjected to and
ERGsacrificed. FourFour
and sacrificed. experimental groups
experimental of eyes
groups werewere
of eyes generated:
gener-
ated:
CTL, CTL,
EVC, EVC,
CTL-MB CTL-MB and EVC-MB.
and EVC-MB.

2.2. Scotopic
2.2. Scotopic Electroretinography
Electroretinography (ERG)
(ERG)
Fifteen days
Fifteen days after
aftersurgery,
surgery,the
thefirst
firstgroup
groupofofrats
rats(n(n= =
1010 per
per group)
group) waswas subjected
subjected to
electroretinography, as described in [30]. Briefly, after an overnight adaptation in thein
to electroretinography, as described in [30]. Briefly, after an overnight adaptation the
dark,
dark,
rats ratsanaesthetized
were were anaesthetized with ketamine/xylazine
with ketamine/xylazine under dim under
red dim red illumination.
illumination. An ophthal-An
ophthalmic solution containing 5% phenylephrine hydrochloride and
mic solution containing 5% phenylephrine hydrochloride and 0.5% tropicamide (Fotor- 0.5% tropicamide
(Fotorretin,
retin, Poen, Buenos
Poen, Buenos Aires, Argentina)
Aires, Argentina) was used wasto used
dilatetothedilate
pupilstheand
pupils and
a local a local
propara-
proparacaine ointment (Poen-caina, Poen, Buenos Aires, Argentina) was applied
caine ointment (Poen-caina, Poen, Buenos Aires, Argentina) was applied over the cornea over the
cornea as a local anaesthetic. Rats were placed facing the stimulus at a distance of 25 cm
as a local anaesthetic. Rats were placed facing the stimulus at a distance of 25 cm in a
in a highly reflective environment. Scotopic electroretinograms (ERGs) were recorded
highly reflective environment. Scotopic electroretinograms (ERGs) were recorded from
from both eyes simultaneously, and 20 responses were collected to flashes of unattenuated
both eyes simultaneously, and 20 responses were collected to flashes of unattenuated
white light (1 m, 1 Hz) from a photic stimulator set at maximum brightness. The registered
white light (1 m, 1 Hz) from a photic stimulator set at maximum brightness. The registered
response was amplified (9 cd s/m2 without filter), filtered (1.5-Hz low-pass filter, 500 Hz
response was amplified (9 cd s/m2 without filter), filtered (1.5-Hz low-pass filter, 500 Hz
high-pass filter, notch-activated), and averaged (Akonic BIO-PC, Buenos Aires, Argentina).
high-pass filter, notch-activated), and averaged (Akonic BIO-PC, Buenos Aires, Argen-
The a-wave was estimated as the difference in amplitude between the recording at onset
tina). The a-wave was estimated as the difference in amplitude between the recording at
and the trough of the negative deflection, and the b-wave amplitude was calculated from
onset and the trough of the negative deflection, and the b-wave amplitude was calculated
Biomedicines 2024, 12, 1983 6 of 15

the trough of the a-wave to the following peak. To calculate oscillatory potentials (OPs), the
same photic stimulator was used with filters of high (300 Hz) and low (100 Hz) frequency.
The amplitudes of the OPs were recorded by using the peak-to-trough method.

2.3. Pattern Electroretinography (PERG)

Fifteen days after surgery, another group of rats (n = 15 per group) was subjected to
pattern electroretinography, as described in [31], with slight modifications. Briefly, rats
were anaesthetized under dim light with an intraperitoneal injection of ketamine/xylazine,
as above. A local proparacaine ointment (Poen-caina, Poen, Buenos Aires, Argentina)
was applied over the cornea. Rats were placed at a 45◦ angle, so one of their eyes was
facing the stimulus at a distance of 20 cm. Transient PERGs were recorded from each eye,
separately. The visual stimulus was generated by commercial software (Akonic BIO-PC,
Buenos Aires, Argentina) and displayed on a CRT monitor (SyncMaster 591s, Samsung,
Suwon-si, South Korea). It consisted of a black and white full 10 × 8 checkboard with a
50% duty cycle that alternated at a temporal frequency of 2 reversals per second (1.00 Hz)
and a spatial frequency of 0.068 cycles per degree. Contrast was maintained at 90%, and
mean luminescence of the projected display was 50 cd/m2 . Duration of the stimulus was
300 milliseconds and results were the average of 100 cycles. N2 wave amplitude was
calculated as the difference between the P1 peak and the following through. N2 peak
latency is the time from the initiation of the stimulus until the N2 through occurs.

2.4. Histology and Morphological Evaluation

After completing the electroretinography, the rats were deeply anaesthetized with an
intraperitoneal injection of ketamine/xylazine and sacrificed by decapitation. Eyes were
enucleated and fixed in 4.0% paraformaldehyde for 48 h at 4 ◦ C, dehydrated, paraffin-
embedded, and sectioned (3.0 µm-thick). Sections were stained with haematoxylin and
eosin (H&E). Before assays, care was taken in selecting anatomically matching areas among
animals for an accurate analysis. To avoid variations in the quantification process, all
images were obtained the same day and under the same light and contrast conditions. The
number of RGCs per 500 µm of retinal length and inner retinal (IR) thickness was calculated
for each experimental group (6 eyes, 7 fields per eye, for a total of 42 fields per group), as
previously reported [32]. The IR is described as the sum of the ganglion cell layer, the inner
limiting layer, and the optic nerve fibre layer, and its thickness usually correlates with the
severity of different retinopathies [33]. Two different regions were evaluated in each retina,
peripheral and central. The distinction between both was performed based on whole-retinal
thickness, as reported [34]. A value of 220 µm was selected as the threshold for dividing
the central retina (>220 µm) from the peripheral retina (<220 µm). Quantification of these
parameters was performed using ImageJ software, version 1.53 g (Wayne Rasband & Co.,
National Institute of Health, Bethesda, MD, USA) in two different areas, peripheral and
central retina, in all eyes.

2.5. Statistical Analysis

All data were analysed with GraphPad Prism, version 10.2.3, software and were
considered statistically significant when p < 0.05. Values are expressed as means ± SEM.
All data sets were evaluated for normality (Shapiro–Wilk) and homoscedasticity (Levene).
Normally distributed data were evaluated by one-way ANOVA followed by the Dunnet’s
(Bonferroni) post hoc test, while data not following a normal distribution were analysed
with the Kruskal–Wallis test followed by the Mann–Whitney U test. In this case, all data
sets followed a normal distribution.

3. Results
3.1. Surgery and Treatment Groups
Seven days post-surgery, half of the rats started intraperitoneal treatment with 2.0 mg/kg
MB, while the other half received the same volume of vehicle (PBS). This design generated
 3. Results
3.1. Surgery and Treatment Groups
Biomedicines 2024, 12, 1983 Seven days post-surgery, half of the rats started intraperitoneal treatment with 2.0
7 of 15
mg/kg MB, while the other half received the same volume of vehicle (PBS). This design
generated four experimental eye groups (Figure 2): (i) sham-operated, injected with vehi-
cle (CTL), (ii) EVC-operated, injected with vehicle (EVC), (iii) sham-operated, injected
four experimental eye groups (Figure 2): (i) sham-operated, injected with vehicle (CTL),
with MB (CTL-MB) and (iv) EVC-operated, injected with MB (EVC-MB).
(ii) EVC-operated, injected with vehicle (EVC), (iii) sham-operated, injected with MB (CTL-
MB) and (iv) EVC-operated, injected with MB (EVC-MB).
3.2. Methylene Blue Restores Electroretinogram Patterns
3.2. Methylene
One day Blue
afterRestores Electroretinogram
finishing the treatments,Patterns
all animals were subjected to electrophysio-
logical
Oneevaluation with onetheoftreatments,
day after finishing two different paradigms:
all animals scotopic electroretinography
were subjected to electrophysiolog-
(ERG, n = 10 per group) or pattern electroretinography (PERG, n = 15 per group). (ERG,
ical evaluation with one of two different paradigms: scotopic electroretinography
n = 10 per group) or pattern electroretinography (PERG, n = 15 per group).
3.2.1. Scotopic Electroretinography
3.2.1. Scotopic
Scotopic ERG
Electroretinography
showed that those eyes subjected to the EVC procedure had a signifi-
cantScotopic ERGreduction
(p < 0.0001) showed thatof those
the a-eyes
andsubjected to the EVCcompared
b-wave amplitude procedure tohad a significant
control sham-
(p < 0.0001) reduction of the a- and b-wave amplitude compared to control
operated eyes (CTL group) (Figure 3A–C). Treatment with MB of sham-operated eyes sham-operated
eyes (CTL group)
(CTL-MB) had no(Figure
impact3A–C). Treatment
on these with MB
parameters of sham-operated
(Figure 3A–C). On theeyes (CTL-MB)
other had
hand, treat-
no impact
ment withonMBthese parameters
of eyes (Figure
subjected 3A–C).
to EVC On the other
(EVC-MB) hand,
resulted in treatment withpreservation
a significant MB of eyes
subjected to EVC
of the a-wave (EVC-MB)
amplitude (p resulted
= 0.0059)inand
a significant preservation
b-wave amplitude (p =of0.0010)
the a-wave
versusamplitude
the EVC
(p = 0.0059) and b-wave
group (Figure 3A–C). amplitude (p = 0.0010) versus the EVC group (Figure 3A–C).

Figure 3. MB prevents changes in the scotopic ERG induced by EVC. (A) Representative electroretino-
grams taken 15 days post-surgery for animals that received either PBS or MB. The red line corresponds
to the right eye, whereas the blue line is the recording of the left eye. (B) Amplitude of the a-wave
in the four experimental groups. (C) Amplitude of the b-wave in the four experimental groups. In
both cases, EVC induced a significant decrease in the a- and b-wave compared to control (CTL),
whereas MB partially or completely prevented it. Each bar represents the mean ± SEM of 10 animals.
One-way ANOVA. Asterisks indicate significant differences: * p < 0.05; ** p < 0.01; **** p < 0.0001.
 retinograms taken 15 days post-surgery for animals that received either PBS or MB. The red line
corresponds to the right eye, whereas the blue line is the recording of the left eye. (B) Amplitude of
the a-wave in the four experimental groups. (C) Amplitude of the b-wave in the four experimental
groups. In both cases, EVC induced a significant decrease in the a- and b-wave compared to control (CTL),
Biomedicines 2024, 12, 1983 whereas MB partially or completely prevented it. Each bar represents the mean ± SEM of 10 animals. 8 of 15
One-way ANOVA. Asterisks indicate significant differences: * p < 0.05; ** p < 0.01; **** p < 0.0001.

3.2.2. Oscillatory
3.2.2. Oscillatory Potentials
Potentials
Similar observations
Similar observations were
weremade
madewhenwhenstudying
studying thethe
OPOPrecordings. Compared
recordings. Comparedto con-
to
trols (CTL), EVC surgery produced a significant (p < 0.0001) loss of complexity
controls (CTL), EVC surgery produced a significant (p < 0.0001) loss of complexity in the in the OP
patterns
OP whereas
patterns MB MB
whereas treatment significantly
treatment (p = (p
significantly 0.0046) preserved
= 0.0046) OP waveform
preserved OP waveformcom-
plexity in a manner undistinguishable from controls. Similarly to the a- and b-waves,
complexity in a manner undistinguishable from controls. Similarly to the a- and b-waves, ap-
plication ofof
application MBMBtoto
sham-operated
sham-operated controls had
controls hadnonoimpact
impact onon
the
theOPs
OPs(Figure
(Figure4A,B).
4A,B).

Figure 4.
Figure 4. MB
MB prevents
prevents changes
changes inin the
the oscillatory
oscillatory potentials
potentials induced
induced by
by EVC.
EVC. (A)
(A)Representative
Representative
oscillatory potentials (OP) of the electroretinograms taken 15 days post-surgery
oscillatory potentials (OP) of the electroretinograms taken 15 days post-surgery for animalsfor animals that
that
received either PBS or MB. The red line corresponds to the right eye whereas the blue
received either PBS or MB. The red line corresponds to the right eye whereas the blue line is the line is the
recording of the left eye. (B) Sum of amplitudes of the OP in the four experimental groups. EVC
recording of the left eye. (B) Sum of amplitudes of the OP in the four experimental groups. EVC
induced a significant decrease in the OP compared to control (CTL), whereas MB treatment pre-
induced a significant decrease in the OP compared to control (CTL), whereas MB treatment prevented
vented it. Each bar represents the mean ± SEM of 10 animals. One-way ANOVA. Asterisks indicate
it. Each bar
significant represents**the
differences: p < mean ± SEM
0.01; **** of 10 animals. One-way ANOVA. Asterisks indicate
p < 0.0001.
significant differences: ** p < 0.01; **** p < 0.0001.
3.2.3. Pattern Electroretinography
3.2.3. Pattern Electroretinography
PERGswere
PERGs werealso
also recorded
recordedto to evaluate
evaluate with
with higher
higher sensitivity
sensitivitythe
theRGCs’
RGCs’ function
functionandand
integrity.Two
integrity. Two main
main parameters
parameters werewere evaluated
evaluated because
because of relevance
of their their relevance for glaucoma
for glaucoma [35,36]:
[35,36]: amplitude and implicit time of the N2 wave (Figure 5A). Eyes
amplitude and implicit time of the N2 wave (Figure 5A). Eyes subjected to EVC surgery subjected to EVC
surgery presented
presented a significant
a significant (p < 0.0001)(p <reduction
0.0001) reduction in N2 amplitude
in N2 amplitude in relationinto
relation to the
the controls
(CTLs). In contrast, treatment of eyes subjected to EVC with MB (EVC-MB) resulted inre-
controls (CTLs). In contrast, treatment of eyes subjected to EVC with MB (EVC-MB) a
sulted in a(psignificant
significant (p < 0.0001)ofpreservation
< 0.0001) preservation N2 amplitude, ofwith
N2 amplitude, withundistinguishable
values that were values that were
undistinguishable
from from theAs
the controls (CTL-MB). controls (CTL-MB).
with regular ERGs,AsMBwith
hadregular
no effectERGs, MB had no effect
on sham-operated eyes
(Figure 5A,B). In the case of N2 implicit time, EVC caused a significant (p < 0.0001)caused
on sham-operated eyes (Figure 5A,B). In the case of N2 implicit time, EVC delay ina this
sig-
nificant (p < 0.0001) delay in this parameter. The application of MB was not
parameter. The application of MB was not able to induce a significant recovery of N2 implicitable to induce
a significant
time recovery of N2 implicit time (Figure 5A,C).
(Figure 5A,C).
cines 2024, Biomedicines
12, x FOR PEER
2024,REVIEW
12, 1983 9 of 15 9 of 15

Figure 5. MB prevents
Figure changes in the PERG
5. MB prevents induced
changes in thebyPERG
EVC. induced
(A) Representative
by EVC. (A) pattern electro-
Representative pattern elec-
retinograms takentroretinograms taken 15 days post-surgery for animals that received either PBSline
15 days post-surgery for animals that received either PBS or MB. The red or MB. The red line
corresponds to the right eye, to
corresponds whereas theeye,
the right bluewhereas
line is the
therecording ofthe
blue line is therecording of Amplitude
left eye. (B) the left eye.of(B) Amplitude of
the N2 wave in the four experimental groups. (C) N2 implicit time in the four experimental groups.
the N2 wave in the four experimental groups. (C) N2 implicit time in the four experimental groups.
EVC induced a significant decrease in the N2 amplitude compared to the control (CTL), whereas it
EVC induced a significant decrease in the N2 amplitude compared to the control (CTL), whereas it
increased the N2 implicit time. MB treatment completely restored N2 amplitude but only partially
increased
restored its implicit the N2 implicit
time. Dotted time. MB
lines indicate treatment completely
the amplitude restored
of the N2 wave. N2 amplitude
Dashed lines show but only partially
the N2 implicit time. Each bar represents the mean ± SEM of 15 animals. One-way ANOVA. Aster- lines show the
restored its implicit time. Dotted lines indicate the amplitude of the N2 wave. Dashed
N2 implicit
isks indicate significant time. Each
differences: ****bar
p < represents
0.0001. the mean ± SEM of 15 animals. One-way ANOVA. Asterisks
indicate significant differences: **** p < 0.0001.
3.3. Methylene Blue Prevents Loss of RGC and IR Thickness
3.3. Methylene Blue Prevents Loss of RGC and IR Thickness
In the histological slides stained with H&E, the number of RGCs and IR thickness in
In the histological slides stained with H&E, the number of RGCs and IR thickness in
the peripheral and central retinal sections were analysed.
the peripheral and central retinal sections were analysed.
3.3.1. Peripheral Retina
3.3.1. Morphology
Peripheral Retina Morphology
At the peripheral
At retina, EVC-operated
the peripheral eyes had a significant
retina, EVC-operated eyes had(p a< significant
0.0001) loss(pof<RCGs
0.0001) loss of RCGs
compared withcompared
the CTL groupwith the CTL group (Figure 6A,B). In contrast, of
(Figure 6A,B). In contrast, MB treatment MBEVC-operated
treatment of EVC-operated
eyes showed aeyes
significant
showed(pa<significant
0.0001) preservation
(p < 0.0001)ofpreservation
RGC count,of although total although
RGC count, values total values
were still lower thanstill
were in lower
the controls
than in(CTL-MB)
the controls(p(CTL-MB)
< 0.001). MB
(p <treatment
0.001). MBdid not modify
treatment did not modify the
the number of number
RGCs inof sham-operated eyes (CTL-MB) (Figure 6A,B).
RGCs in sham-operated eyes (CTL-MB) (Figure 6A,B).
Biomedicines 2024, 12,
Biomedicines 1983
2024, 12, x FOR PEER REVIEW 10 of 1510 of 15

Biomedicines 2024, 12, x FOR PEER REVIEW 10 of 15

Figure
Figure 6. Death
6. Death of of RGCs
RGCs andinner
and innerretina
retinathickening
thickening are areprevented
preventedbybyMB MB treatment
treatment in the periph-
in the peripheral
eral retina. Representative histological images of the peripheral retina of animals of the 4 experi-
retina. Representative
mental groups, takenhistological
15 days afterimages
surgery,ofandthestained
peripheral retina of animals (A).
with hematoxylin–eosin of the 4 experimental
Three layers
groups,
of thetaken
Figure retina 15
6. Deatharedays
of after
RGCs
labelled and
in surgery,
theinner and
retina
pictures stained
reference:with
forthickening are
outer hematoxylin–eosin
prevented by MB
nuclear layer (A).
innerThree
treatment
(ONL), in layers
layer of the
the periph-
nuclear
eral
retina retina.
(INL),
are and Representative
ganglion
labelled in the histological
cellpictures
layer (GCL). images
Scale bar =of
for reference: 50the
outerµm.peripheral retina
Quantification
nuclear layer of the
of
(ONL), animals
number
inner ofnuclear
the 4 experi-
of RGCs (B) (INL),
layer
mental groups, taken 15shown
days after surgery, and stained withthe hematoxylin–eosin of all(A). Three(6layers
andand IR thickness
ganglion (C) are
cell layer as histograms.
(GCL). Scale bar = 50Bars
µm.represent
Quantificationmeanof ± SEM
the number samples
of RGCs eyes
(B) and
of
perthe retinaOne-way
group). are labelled in the followed
ANOVA pictures for
by reference:
Bonferroniouter
post nuclear
hoc test.layer (ONL),
Asterisks inner nuclear
represent layer
statistically
IR thickness
(INL), (C) are
and differences:
significant
shown
ganglion cell***layeras histograms.
(GCL).****
p < 0.001; Scale Bars
p < bar
0.0001.
±
= 50 µm. Quantification of the number of RGCs (B)(6 eyes
represent the mean SEM of all samples
per and
group). One-way
IR thickness (C)ANOVA
are shownfollowed by Bonferroni
as histograms. postthe
Bars represent hocmean
test.±Asterisks
SEM of allrepresent
samples (6statistically
eyes
per group).
significant One-way
differences:
Regarding ANOVA
IR thickness, followed
*** p < 0.001;there****by
p <Bonferroni
was 0.0001.
also post hoc(p
a significant test. Asterisks
< 0.0001) represent
thinning ofstatistically
this layer
significant differences: *** p < 0.001; **** p < 0.0001.
generated by EVC surgery (Figure 6A,C). MB treatment (EVC-MB group) completely pre-
Regarding
vented IR thickness,
(p < 0.0001) there was
this modification and also
wasa indistinguishable
significant (p < 0.0001)from the thinning
controlsof this layer
(CTL-
Regarding IR thickness, there was also a significant (p < 0.0001) thinning of this layer
generated
MB). As with by EVC othersurgery
parameters, (Figure
MB had 6A,C). MBon
no effect treatment
sham-operated(EVC-MB group)6A,C).
eyes (Figure completely
generated by EVC surgery (Figure 6A,C). MB treatment (EVC-MB group) completely pre-
prevented (p < 0.0001) this modification and was indistinguishable from the controls (CTL-
vented (p < 0.0001) this modification and was indistinguishable from the controls (CTL-
MB).3.3.2.
As Central
with other Retina Morphology
parameters, MB had no effect on sham-operated eyes (Figure 6A,C).
MB). As with other parameters, MB had no effect on sham-operated eyes (Figure 6A,C).
The morphological changes observed in the central retina were similar to those de-
3.3.2. Central
scribed
3.3.2. in the
Central
Retina Morphology
peripheral
Retina retina, but with some differences. Here, there was also a clear
Morphology
reduction
The (p < 0.0001) in changes
morphological both RGCobserved
counts andinIRthe thickness
central due to EVC surgery (Figure
The morphological changes observed in the central retinaretina were
were similar similar
to those de- to those
7A–C). Both
described in parameters
the peripheral were significantly
retina, but (p ≤ some
with 0.0001)differences.
preserved by Here, treatment there withwas MBalso a
scribed in the peripheral retina, but with some differences. Here, there was also a clear
but, in this case, both parameters were lower than the controls (CTL-MB) (p < 0.0001). As
clear reduction
reduction (p < (p < 0.0001)
0.0001) in bothinRGC bothcounts
RGCand counts and IR due
IR thickness thickness
to EVCdue surgery to EVC(Figure surgery
in the peripheral retina, the application of MB to sham-operated eyes had no effects (Fig-
(Figure
7A–C). 7A–C).
Both Both parameters
parameters were significantly
were significantly (p ≤preserved
(p ≤ 0.0001) 0.0001) preserved
by treatment by treatment
with MB with
ure 7A–C).
MBbut,but,ininthisthiscase,
case,both
bothparameters
parameters werewerelowerlower
than the
thancontrols (CTL-MB)
the controls (p < 0.0001).
(CTL-MB) (p <As 0.0001).
As in
in the
theperipheral
peripheral retina, the the
retina, application
applicationof MBof toMB
sham-operated eyes had eyes
to sham-operated no effects
had (Fig-
no effects
ure 7A–C).
(Figure 7A–C).

Figure 7. Death of RGCs and inner retina thickening are prevented by MB treatment in the central
retina. Representative histological images of the central retina of animals of the 4 experimental
groups, taken 15 days after surgery, and stained with hematoxylin–eosin (A). Three layers of the
Figure 7. Death of RGCs and inner retina thickening are prevented by MB treatment in the central
Figure 7. Death
retina. of RGCshistological
Representative and inner images
retina thickening areretina
of the central prevented by MB
of animals treatment
of the in the central
4 experimental
retina. Representative
groups, taken 15 dayshistological
after surgery,images of the
and stained central
with retina of animals
hematoxylin–eosin of the
(A). Three 4 experimental
layers of the
groups, taken 15 days after surgery, and stained with hematoxylin–eosin (A). Three layers of the
retina are labelled in the pictures for reference: outer nuclear layer (ONL), inner nuclear layer (INL),
Biomedicines 2024, 12, 1983 11 of 15

and ganglion cell layer (GCL). Scale bar = 50 µm. Quantification of the number of RGCs (B) and
IR thickness (C) are shown as histograms. Bars represent the mean ± SEM of all samples (6 eyes
per group). One-way ANOVA followed by Bonferroni post hoc test. Asterisks represent statistically
significant differences: **** p < 0.0001.

4. Discussion
In this study, we have demonstrated that MB treatment can be effective in preventing
physiological and morphological hallmarks of optic neuropathy in a rat model of ocular
hypertension, which faithfully recapitulates human POAG, the most common clinical
presentation of glaucoma [3]. This study follows our previous work, where we showed
that MB treatment had a beneficial impact in the retinal integrity of newborns subjected to
perinatal asphyxia [28,29]. Based on these previous findings, we decided to apply this novel
neuroprotective strategy to a model of hypertensive glaucomatous neuropathy induced by
EVC, generating data that may change the current paradigm of glaucoma treatment.
Interestingly, MB exhibits a high safety profile [24,37] and is approved for clinical use
as an antidote of poison-induced methemoglobinemia [38], in norepinephrine refractory
hypotension [39], and for the surgical management of hyperparathyroidism [37], among
other applications. MB is on the World Health Organization’s List of Essential Medicines,
which gives the most effective and safe medicines needed in a health system [40]. The
only known incompatibility with MB is the use of serotoninergic drugs [41], so special care
should be taken with glaucoma patients who are treated with antidepressants.
Scotopic ERG findings showed that EVC resulted in a very significant reduction in
the amplitude of both the a- and b-waves, which represent the electrical recordings of
the photoreceptors’ and the bipolar/Müller glia cells’ activity, respectively [42]. Even
though there is no direct damage to bipolar cells and Müller glia in glaucoma, their
electrical activity is most likely affected by RGC compromise, therefore resulting in b-wave
amplitude reduction. The implication of the RGCs is the expected electrophysiological
hallmark of glaucoma, given the compression of RGCs’ axons at the lamina cribosa due
to ocular hypertension (OHT). This compression produces cellular stress by axoplasmic
flux blockade, decrease in transport of neurotrophic factors to the soma and, eventually,
neuronal cell death [2,43,44]. The administration of MB resulted in a significant restoration
of the ERG’s waveform and amplitude for both waves. These findings account for inner
retina neuroprotection, especially of the RGCs, which are the cells most affected by this
pathology. This is also in agreement with the morphological results, which showed a
significant preservation of the number of RGCs in the retinas treated with MB. As for the
a-wave findings, no morphological alterations were observed at the photoreceptor layer, as
was expected of an OHT model. Therefore, the electrophysiological changes in the a-wave
are most likely a result of retrograde electrical current influences from the RGCs that do
not result in photoreceptor structural damage, in a similar manner to that described for the
b-wave and bipolar cells. This phenomenon has already been described in other models of
retinopathy [29,45,46].
In addition, scotopic OPs were also significantly altered by EVC. Scotopic OPs are
a high-frequency low-voltage component of the ERG, which correlates tightly with the
b-wave, and may be seen as slight indentations in its ascending limb. However, their origin
is slightly different from the cells that produce the b-wave, and depends on the electrical
activity of bipolar and amacrine cells, instead [47,48]. It is well known that OPs constitute
a very sensitive parameter of vascular dysfunction, including that produced by hypoxia,
in the retina, and they even precede a- and b-wave alterations [47,49,50]. As for the exact
significance of OP modifications in glaucoma, the verdict is still inconclusive, though these
alterations have been previously described [51–53]. In our experiment, we found that the
OPs in the EVC group had a significantly lower complexity in relation to the controls,
something that usually indicates the presence of some retinopathy/neuropathy [32,54].
The distortion of the OP pattern found in glaucomatous eyes could be a consequence of
the initial damage to the RGCs, which would result in a synaptic dysfunction at the inner
Biomedicines 2024, 12, 1983 12 of 15

plexiform layer, where RGCs connect with bipolar and amacrine cells. Furthermore, due to
hemodynamic alterations produced by the IOP, areas of ischemia/hypoxia may occur in
the inner retina, thus generating OP distortion. Interestingly, this distortion was completely
prevented by the treatment with MB, suggesting a clear neuroprotective effect for this drug.
To further characterize the effects of our glaucoma model and the posterior MB
effects in the RGCs, we performed PERG analyses, which have been described as the
most sensitive method to evaluate RGC function [31], even before significant losses of
RGCs and vision field are evident [35,36]. N2 wave (equivalent to human PERG N95
wave) amplitude and its implicit time were quantified. This wave is originated exclusively
from RGCs [55] and, chronologically, is the first electrophysiological alteration found in
glaucoma pathophysiology. As expected, EVC eyes showed a significant reduction in N2
amplitude compared to controls, whereas N2 implicit time was significantly prolonged.
Increased N2 peak latency has been associated with RCG dysfunction, even before cell
death occurs [56]. On the other hand, MB treatment showed a significant preservation of
N2 amplitude in relation to EVC, with values identical to controls, thus confirming the
great potential of this drug as a neuroprotective agent. Regarding N2 implicit time, MB
did not produce a significant recovery. Nevertheless, the N2 implicit time of MB-treated
EVC eyes was also indistinguishable from MB-treated controls. These findings suggest that
MB significantly prevented RGC death, but some of these viable cells may still have signs
(slightly increased N2 implicit time) of slow conductance and cell dysfunction, probably
due to OHT-mediated cellular stress.
Histological evaluation of H&E retinal sections showed a significant loss of RGC
counts and IR thickness in the EVC group in comparison to controls. These morphological
hallmarks coincided with the electrophysiological findings, for both ERG and PERG. Treat-
ment with MB translated into a highly significant restoration of most of these parameters
supporting the relevance of adding this drug to the current treatments for glaucoma.
This study has some limitations. First, this work was designed as a proof-of-concept,
so experiments were carried out exclusively in male rats to reduce potential variability
due to sex and hormonal influences. In humans, men have a higher burden of glaucoma
than women [57] and there is growing evidence that sex hormones play a role in the
pathophysiology of glaucoma [58]. Future studies will show whether these hormones have
any influence in the efficacy of the MB treatment. In addition, MB is a very soluble drug.
This characteristic has advantages and disadvantages. The main advantage is shown in
our study, where, by using an intraperitoneal injection, we were able to influence retinal
pathophysiology, demonstrating that MB can easily cross the blood–ocular barrier. But
this behaviour also poses a clear pharmacokinetic disadvantage, namely that MB will
remain in the eyeball only for short periods of time. Therefore, if we want to maintain a
chronic treatment with MB, very frequent injections (twice a day in our experiment) will be
necessary. Since glaucoma is a chronic disease that often requires lifelong management, we
are actively pursuing new strategies to increase MB´s half-life in the eye and investigating
alternative administration routes, including topic application.

5. Conclusions
In summary, we have shown MB to be an effective treatment for reducing electrophysi-
ological and morphological alterations generated by ocular hypertension and glaucomatous
retinal damage. Due to its high safety profile, this drug could therefore represent a new
pharmacologic strategy to prevent vision losses in glaucoma patients. Nevertheless, fur-
ther research is needed to solve the current pharmacokinetic obstacles that this molecule
represents for the treatment of a chronic condition such as glaucoma.

Author Contributions: Conceptualization, C.F.L., A.M. and M.R.-F.; funding acquisition, A.M.;
investigation, R.N., N.S.C., J.C.F., R.P., Á.P.-S., M.B. and J.J.L.-C.; supervision, C.F.L., A.M. and M.R.-F.;
writing—original draft, R.N., A.M. and M.R.-F.; writing—review and editing, R.N., N.S.C., J.C.F.,
R.P., Á.P.-S., M.B., J.J.L.-C. and C.F.L. All authors have read and agreed to the published version of
the manuscript.
Biomedicines 2024, 12, 1983 13 of 15

Funding: This research was funded by UBACyT; Grants 20020220100061BA (to JJL) and 20020190100284BA
(to CFL), and by Fundación Rioja Salud (ONCO1) (to AM).
Institutional Review Board Statement: The animal study protocol was approved by the Ethical
Committee of CICUAL (Comité Institucional para el Uso y Cuidado de Animales de Laboratorio),
Facultad de Medicina, Universidad de Buenos Aires, Argentina. Resolution number RESCD-2023-
1408-E-UBA-DCT#FMED.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusion of this article will be made
available by the authors, without undue reservation.
Acknowledgments: We want to acknowledge Andrea Pecile and Marianela Ceol-Retamal for excellent
assistance at the animal facility, Ing., Lisandro Antón for electroretinography set-up, and Alicia Araoz
for histological assistance.
Conflicts of Interest: The authors declare no conflicts of interest.

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