Accepted Manuscript

Low-level laser therapy modulates demyelination in mice

Katherine Chuere Nunes Duarte, Thaís Torres Soares, AngelaMaria Paiva Magri, Lívia Assis Garcia, Luciana Le Sueur Maluf,Ana Cláudia Muniz Renno, Gláucia Monteiro de Castro

PII: S1011-1344(18)30361-0DOI: doi:10.1016/j.jphotobiol.2018.09.024Reference: JPB 11365

To appear in: Journal of Photochemistry & Photobiology, B: Biology

Received date: 6 April 2018Revised date: 28 August 2018Accepted date: 25 September 2018

Please cite this article as: Katherine Chuere Nunes Duarte, Thaís Torres Soares, AngelaMaria Paiva Magri, Lívia Assis Garcia, Luciana Le Sueur Maluf, Ana Cláudia MunizRenno, Gláucia Monteiro de Castro , Low-level laser therapy modulates demyelination inmice. Jpb (2018), doi:10.1016/j.jphotobiol.2018.09.024

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Low-Level Laser Therapy modulates demyelination in mice

Katherine Chuere Nunes Duarte1,2, Thaís Torres Soares1,2, Angela Maria Paiva Magri1,2, Lívia

Assis Garcia1,2, Luciana Le Sueur Maluf1,2, Ana Cláudia Muniz Renno1,2,3, Gláucia Monteiro de

Castro1,2

1) Programa Interdisciplinar em Ciências da Saúde - Universidade Federal de São Paulo -

UNIFESP – Av. Ana Costa, 95 – Santos-SP –Brazil– 11060-001

2) Departamento de Biociências - Universidade Federal de São Paulo - UNIFESP – Rua Silva

Jardim, 136 – Santos-SP –Brazil– 11015-020

3) Programa de Bioprodutos e Bioprocessos - Universidade Federal de São Paulo - UNIFESP

– Av. Ana Costa, 95 – Santos-SP –Brazil– 11060-001

Corresponding author

Gláucia Monteiro de Castro

Rua Silva Jardim, 136 – Sala 323 - Santos-SP, Brazil. 11015-020

Tel.: +55 13 3229 0180

E-mail: glaucia.castro@unifesp.br

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Abstract

There are no effective therapies for remyelination. Low-level laser therapy (LLLT) has been found

advantageous in neurogenesis promotion, cell death prevention, and modulation of inflammation in

central and peripheral nervous system models. The purpose of this study was to analyse LLLT effects on

cuprizone-induced demyelination. Mice were randomly distributed into three groups: Control Laser (CTL),

Cuprizone (CPZ), and Cuprizone Laser (CPZL). Mice from CPZ and CPZL groups were exposed to a

0.2% cuprizone oral diet for four complete weeks. Six sessions of transcranial laser irradiation were

applied on three consecutive days, during the third and fourth weeks, with parameters of 36 J/cm2, 50

mW, 0.028 cm2 spot area, continuous wave, 1 J, 20 seconds, 1.78 W/cm

2 in a single point equidistant

between the eyes and ears of CTL and CPZL mice. Motor coordination was assessed by the rotarod test.

Twenty-four hours after the last laser session, all animals were euthanized, and brains were extracted.

Serum was obtained for lactate dehydrogenase toxicity testing. Histomorphological analyses consisted of

Luxol Fast Blue staining and immunohistochemistry. The results showed that laser-treated animals

presented motor performance improvement, attenuation of demyelination, increased number of

oligodendrocyte precursor cells, modulated microglial and astrocytes activation, and a milder toxicity by

cuprizone. Although further studies are required, it is suggested that LLLT represents a feasible therapy

for demyelinating diseases.

INTRODUCTION

Central nervous system (CNS) demyelination is characterized by oligodendrocyte degeneration.

Loss of myelin sheath impairs nerve impulse transmission, originating disability symptoms such as motor,

sensory or cognitive abnormalities [1]. Demyelination can be triggered by primary oligodendrocyte death,

as in multiple sclerosis and some leukodystrophies.

It is proposed that the lesion caused by demyelination acts as part of a vicious circle, in which

autoimmune attacks or enzymatic defects result in oligodendrocytes’ death, formation of myelin debris

and white matter degeneration, sustaining the activation of microglia and astrocytes. These cells, in turn,

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express pro-inflammatory cytokines, enhancing inflammation and further harming oligodendrocytes and

neurons [2, 3, 4].

Although the hostile environment created by the autoimmune attack, deposition of extracellular

matrix components, and accumulation of myelin debris affects remyelination, the inflammatory

environment is important to promote it [5]. After a period of demyelination, activated astrocytes and

microglia express neurotrophic factors that stimulate oligodendrogenesis [6, 7].

Cuprizone is a classic demyelinating model, although the mechanism is not completely clear

[8,9,10, 11]. It is believed that cuprizone is implicated in mitochondrial impairment, leading to

oligodendrocyte apoptosis by energy failure, endoplasmic reticulum stress and reactive oxygen species

generation [12, 13]. After four weeks of cuprizone exposure, severe demyelination is detected, especially

in corpus callosum, associated with activation of microglia and astrocytes [11, 14]. During chronic

treatment with cuprizone, following severe demyelination phase, Oligodendrocyte Precursor Cells (OPCs)

can proliferate. However, the differentiation is inhibited and the remyelination process is impaired [15].

There is no effective treatment for demyelinating diseases and this motivates the search for

alternative therapies that could improve the functional recovery [16]. Additionally, low-level laser therapy

(LLLT) has been shown to be positive in various medical applications such as musculoskeletal lesions,

dermatitis, ulcers, rheumatological disorders, major depressive disorders, and pain relief [17, 18, 19, 20,

21, 22].

Previous studies have shown the effect of LLLT on the nervous system. In the peripheral nervous

system (PNS), LLLT improved clinical signs in rats submitted to sciatic nerve injury [23,24, 25]. In the

CNS, Rochkind and cols. demonstrated neuronal sprouting and repair in spinal cord lesions in mice

engrafted with embryonic nerve cells and treated with LLLT [26]. Xuan et al. observed in a traumatic brain

injury (TBI) model that LLLT treatment stimulated cell proliferation, correlating with increased brain-

derived neurotrophic factor (BDNF) [27, 28]. In ischemic stroke model, LLLT upregulated anti -apoptotic

and decreased pro-apoptotic marker expression in mice, improved clinical outcome, and enhanced

neurogenesis [29, 30]. In vitro, LLLT has been shown to reduce reactive oxygen species (ROS) in

cultured neurons, prevent oxidative stress and modulate the inflammatory process [31, 32, 33]. Based on

previous studies, we hypothesised that LLLT could attenuate demyelination induced by cuprizone.

In the present study, we evaluated the LLLT effects on demyelination induced by cuprizone. Our results

showed that LLLT can induce OPCs proliferation associated with a reduction in the microglia and

astrocytes density.

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MATERIALS AND METHODS

All procedures involving animal manipulation in this protocol were approved by the Animal Experimental

Ethics Committee of the Universidade Federal de São Paulo (9018200415).

Animals and cuprizone administration

Male C57BL/6 mice, aged 7 weeks (Centro de Desenvolvimento de Modelos Experimentais para

Medicina e Biologia - CEDEME) were acclimated for 1 week before the experiment. The animals were

maintained in the care of Campus Baixada Santista – Universidade Federal de São Paulo, in a light/dark

cycle (12/12h) and constant temperature (22±2ºC). Animals were randomly distributed into Control Laser

(CTL), Cuprizone (CPZ) and Cuprizone Laser (CPZL) groups. The animals from the CPZ and CPZL

groups were fed with 0.2% (w/w) cuprizone (bis(cyclohexanone)oxaldihydrazone – Sigma–Aldrich, St.

Louis, MO, USA) mixed in ground chow, for 4 weeks. The control group was fed with the same ground

chow, without the cuprizone addition.

Transcranial Low-level laser therapy

The laser was applied on 3 consecutive days during the third week and repeated during the fourth week

of cuprizone treatment, totalising 6 sessions in both CTL and CPZL groups. Each session consisted of a

low energy Ga-Al-As (Gallium-aluminium-arsenide) laser, model Photon Laser (DMC Equipamentos

Ltda®), 808 nm wavelength of infrared light with an energy density of 36 J/cm2, average radiant power 50

mW, 0.028 cm2 spot area at target, continuous wave, energy per pulse 1J, irradiance at target 1.78

W/cm2 in a single point equidistant between the eyes and ears of the animal, lasting 20 seconds, an

experienced manipulator, familiar to the animals, gently held the ears and the body for the laser

application without causing any stress [28].

Table 1. Low-Level Laser Therapy Parameters [34]

Device Information Irradiation Parameters Treatment Parameters

Manufacturer: DMC Equipamentos® Centre wavelength: 808nm Beam spot size at target: 0.028cm2

Model: Photon Laser II Spectral bandwidth: +10nm Irradiance at target: 1.78mW/cm2

Year of manufacture: 2012 Operating mode: Continuous wave Exposure duration: 20sec

Number of emitters: 1 Average radiant power: 50mW Radiant exposure: 36J/cm2

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Emitter type: GaAlAs Polarization: Linear Radiant energy [J]: 1J

Spatial distribution of emitters: Diode laser Irradiance at aperture: 1.78/cm2 Nº of points irradiated: 1

Beam delivery system: Hand-held probe Beam divergence: 0.45 rad ± 0,03 Area irradiated: 0,028cm2

Beam shape: Circular Application technique: Skin contact

Number and frequency of

treatment sessions

1x daily for 3 days

in the third week

and fourth week of

cuprizone

administration,

totalizing 6

sessions

Total radiant energy: 6J

Accelerated rotarod test – motor coordination

Motor coordination was assessed by accelerated rotarod test (Insight, EFF-411) on the 1st and 29

th day of

the experiment. Before the test, all animals were habituated on the same day at constant velocity of 4

revolutions per minute (rpm) for 5 minutes. After a resting period of 5 minutes, accelerated protocol was

performed at 4 to 12 rpm for 5 minutes. If the mice fell from the cylinder, the time was paused until its

replacement and then the test continued. Latency and number of falls were registered.

Cuprizone toxicity test

Blood samples were obtained by cardiac puncture before perfusion. The blood was maintained at room

temperature for 30 minutes, centrifuged at 1,500 rpm for 20 minutes, and serums were collected. The

serum concentration of lactate dehydrogenase was assessed by Lactate Dehydrogenase Assay Kit

(Abcam, Cambridge, MA – U.S.A.) following manufacturer’s instructions.

Histology and immunohistochemistry

Twenty-four hours after the final laser application (29th

experiment day), the mice were deeply

anesthetised and perfused transcardially with phosphate-buffered saline (PBS) followed by 4%

paraformaldehyde in PBS. After dissection, tissue samples were post-fixed in 4% paraformaldehyde in

PBS for 2h at 4◦C, cryoprotected in 30% sucrose solution, embedded in Tissue-Tek O.C.T. compound

(Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands), frozen on dry ice, and stored at

80◦C. Sections of 12 µm were obtained by cryostat, mounted onto slides, and allowed to dry.

Demyelination was detected using Luxol Fast Blue (LFB) staining: The sections were immersed in

chloroform/ethanol (1:1- Merck, Darmstadt, Germany) to remove lipids, and afterwards were incubated in

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LFB staining solution (1% LFB in 95% ethanol with 0.5% acetic acid) at 56◦C overnight. Sections were

then differentiated in 0.5% lithium carbonate solution for 30s, counterstained with cresyl violet,

dehydrated in an ethanol series, diaphanized in xylene, and then mounted in Entellan (Merck, Darmstadt,

Germany). For immunostaining, antigen retrieval was performed by incubating slides in 10 mM sodium

citrate, pH6, for 5 min at 95◦C and then for 30 min at room temperature. Slides were then incubated for 1h

at room temperature in blocking solution (5% normal donkey serum, 0.3% TritonX-100 in PBS). Primary

antibody was added to the blocking solution which was then incubated overnight at 4◦C. The following

primary antibodies were used: myelin detection: mouse anti-MBP (1:200 – Millipore, Watford, UK);

astrocytes: rabbit anti-GFAP (1:200); microglia: goat IBA-1 (1:200); OPCs proliferation: mouse anti-

PDGF-beta receptor (1:200) and rabbit anti-Ki67 (1:300); oligodendrocyte lineage: goat anti-OLIG2 (1:300

- Abcam, Cambridge, MA – U.S.A.). The slides were incubated in the following secondary antibodies

(1:400): Alexa Fluor 564 donkey anti-goat, Alexa Fluor 488 and 564 donkey anti-rabbit, Alexa Fluor 488

and 564 donkey anti-mouse (Abcam, Cambridge, MA, U.S.A.) with DAPI (1:1000 4 -’,6-diamidino-2-

phenylindole – Sigma-Aldrich, St. Louis, MO, USA). Coverslips were mounted in FluorSaveTM

(Calbiochem® – U.S.A.). Slides were photographed using an AxioVision fluorescence microscope (Carl

Zeiss, Gottingen, Germany).

Cell semi-quantification

Aiming to avoid any inconsistency, all the measurements were made on corpus callosum. To analyse

myelin density, images were evaluated by randomly placing a frame (100µm x 100µm) and measuring the

optical densities (OD) of LFB stain for the semi-quantification of the intensity of myelin. A higher OD

indicates a higher intensity/transmittance of LFB staining, which is inversely proportional to the myelin

density/amount. The Control Group was taken as a normality parameter and the medium density

considered as 100%, since the myelin was intact. The percentage of myelin density in the CPZ and CPZL

groups was calculated [34]. The immunohistochemistry analysis was conducted by randomly placing

three frames on the corpus callosum and measuring the ODs to semi-quantify MBP and the intensity of

microglia and astrocytes. The counting of immunolabelled positive cells was done by randomly

positioning three frames (10mm2) in different places on corpus callosum (figure 1i) and counting the

positive cells inside these frames, including those touching the superior and left side but excluding the

right and inferior sides. We used ImageJ v.1.48 software (U.S. National Institutes of Health, Bethesda,

Maryland, USA – available as freeware from http://rsbweb.nih.gov/ij/).

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Statistical analysis

All results were calculated as mean values ± SEM. The data were submitted to Kolmogorov-Smirnov

normality test and the results were analysed using GraphPad Prism software version 6. Two-way analysis

of variance (ANOVA) measurements followed by Tukey’s post-hoc test compared each animal’s weight.

Food intake control, rotarod test, LFB, immunohistochemistry, and toxicity test were evaluated through

non-parametric Kruskal-Wallis test followed by multiple comparison Dunn’s test. Statistical values of

p<0.05 were considered significant.

RESULTS

Body weight and chow intake

At the beginning of the experiment, all groups presented similar body weight. However, at the end of the

experimental period, CTL group exhibited a weight gain of 5.1% (22.7±0.9 g), whereas mice fed with

cuprizone exhibited significant weight loss. CPZ group lost 10.7% (19.2±0.4 g; p=0.0006) and CPZL

group lost 8.6% (19.2±0.7 g; p=0.02555; F=1.822), compared with CTL group. No statistical difference in

chow intake was detected in either group (p=0.9364; H=0.1314).

Motor coordination

Motor coordination was assessed by accelerated rotarod test. Before demyelination induction, all animals

exhibited similar latencies (Fig.1a - p=0.9240; n=6 per group) and falling number (Fig. 1b - CTL=0.7±0.2;

CPZ=0.8±0.2; CPZL=0.6±0.2; p=0.8679). After cuprizone exposure, CPZ group showed lower

permanence on the cylinder, with statistical significance at 11rpm in comparison to CTL group (p=0.02).

CPZL group exhibited a similar latency to CTL group. Corroborating latency data, animals from CPZ

group showed the highest number of falls (p<0.05), whereas mice from CPZL group fell less and were

statistically similarly to CTL group (CTL=0.5±0.2; CPZ=0.9±0.1; CPZL=0.8±0.2; p=0.0439).

Toxicity test – serum Lactate Dehydrogenase (LDH) concentration

A colorimetric assay was used investigate the toxicity levels and tissue damage by measuring serum

LDH. In CTL serum, the LDH level was 3,152mU/mL ±158; n=5. A significant increase in LDH

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concentration was seen in CPZ serum compared to CTL (5,624mU/mL ±77; n=6; p=0.0107). An

intermediate LDH level was detected in CPZL serum that was not statistically different from CTL or CPZ

group (5,233mU/mL ±532; n=6; Figure 2).

Demyelination

Demyelination induced by cuprizone was indicated by Luxol Fast Blue (LFB) staining, which is specific to

lipids, and immunohistochemistry was used to detect Myelin Basic Protein (MBP). Corpus callosum from

CTL group exhibited homogeneous distribution of myelin, showing tissue integrity and preserved

cellularity of nervous tissue (figure 3a). In CPZ group, corpus callosum demonstrated multiple pale areas

associated with less intense LFB staining, tissue fragility, and myelin degeneration (figure 3b). LFB

staining observed in corpus callosum from CPZL group suggests less myelin damage than CPZ mice

(figure 3c). Myelin Basic Protein (MBP) immunostaining results were also uniform in corpus callosum from

CTL group (figure 3d). As observed in LFB staining, after cuprizone exposure, MBP immunolabelling was

lower in CPZ group (figure 3e). CPZL group corpus callosum areas exhibited stronger MBP labelling

(figure 3f).

A semi-quantification of the myelin intensity was carried out; a higher optical density (OD) indicates higher

intensity/transmittance and is represented by a lower LFB staining intensity. In comparison to the CTL

group, CPZ animals exhibited a significant decrease in myelin density (79.1±4%; p=0.0275; n=5) and

CPZL group demonstrated intermediate density of LFB stain (91±7.5%; n=4; p=0.3429; figure 3g).

Consistent with the observations of LFB staining, optical density analysis of MBP intensity in CPZ group

was significantly lower than CTL group (19.7±0.9%; p<0.0001; n=5), whereas intensity in CPZL group

was significantly higher compared to CPZ group (50.7±1.2%; p<0.001; n=6; figure 3h).

Oligodendrocyte lineage cells

Olig2 immunostaining was used to quantify positive cells from oligodendrocyte lineage cells in corpus

callosum. A similar number of Olig2 positive cells (Olig2+ - figure 4) were observed in CTL group corpus

callosum (n=6; CTL=2,500±40 cells/mm2) and CPZ (n=5; 2,700±90 cells/mm

2). However, a significantly

higher number was found in the CPZL corpus callosum group (n=6; 3,000±80 cells/mm2; p<0.001).

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The Platelet Derived Growth Factor-beta receptor (PDGFβR) is a surface marker of OPCs, and Ki67 is a

transcription factor expressed during cell proliferation (figure 5). Thus, OPCs in proliferation were

analysed by co-localization and Ki67 immunostaining. There were few positive PDGFβR/Ki67 cells in the

CTL group (image a; n=5; 100±10 cells/mm2), while in CPZ group, a significant increase in the number of

these cells was found (image b; n=5; 1,000±70 cells/mm2; p<0.001). However, the count was even higher

in the CPZL group, compared to CTL and CPZ groups (image c; n=6; 2,100±70 cells/mm2; p<0.0001).

Microglia and Astrocytes

The inflammatory process, triggered by cuprizone-induced demyelination, was analysed by means of

IBA-1 and Glial Fibrillary Acidic Protein (GFAP) immunohistochemistry to detect microglia and astrocytes

respectively, for semi-quantitative analyses. The perimeter of these cells was measured as an indication

of activation. In CTL group, IBA-1+ cells (figure 6a) exhibited small elliptical cell bodies and ramified

cytoplasmic projections morphology, suggestive of quiescent cells. In the CPZ and CPZL groups, most of

the IBA-1+ cells (figure 6, images b and c) showed round shape, increased cell size and retracted

ramifications, suggestive of activated microglia. The semi-quantification of IBA-1+ cells in CTL group

exhibited few microglia dispersed through corpus callosum (500±40 cells/mm2; n=6). CPZ group showed

significantly higher number of microglia (5,000±130 cells/mm2; n=5) in comparison to CTL and CPZL

groups (2,300±80 cells/mm2; n=6 in CPZL; p<0.001; figure 6, graph d). The perimeter analyses also

showed significantly smaller perimeter in CTL group (48±2.9μm; p<0.0001) and CPZL group (52±2.4μm)

in comparison to CPZ group (65±2.7μm, figure 6 e). Due to the higher number of IBA-1+ cells with greater

perimeters in corpus callosum of CPZ animals, the fluorescence intensity immunostaining was higher in

this group in relation to CTL group (p<0.0001). Conversely, the intensity in the CPZL was decreased

compared to CPZ group, due to diminished quantity and perimeter of IBA-1 positive cells (CTL=2±0.2

pixels; CPZ=62±1.3 pixels; CPZL=42±0.8 pixels; p<0.05; figure 6, graph f). The corpus callosum of CTL

animals (figure 6, image g) presented fine processes of GFAP distributed uniformly. Once mice were

exposed to cuprizone, these cells changed to an activated morphology, showing thicker branches and

increased size, observed in CPZ and CPZL groups (figure 6, images h and i). To confirm these

observations, semi-quantitative analyses were performed (figure 6, graph j). The CTL group (1,300±40

cells/mm2; n=6) exhibited significantly fewer GFAP+ cells in comparison to CPZ (3,600±130 cells/mm

2;

n=5; p<0.0001) and CPZL groups (2,500±120 cells/mm2; n=6; p<0.05). After the activation, the

expression of GFAP increased, reflected in the size of the activated astrocytes. The medium perimeter of

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CTL group was 86±3μm (figure 6, graph k), significantly smaller than CPZ group (200±5.9μm; p<0.0001)

and CPZL group (163±4.5 μm; p<0.0001). In addition, CPZ group exhibited bigger perimeters in

comparison with CPZL (p<0.05). Consistent with the previously results, the fluorescence intensity (figure

6, graph l) was significantly reduced in CTL (8±0.5 pixels; p<0.0001) and CPZL (33±0.7 pixels; p<0.001)

groups in comparison with the CPZ group (66±0.6 pixels).

DISCUSSION

This study aimed to evaluate LLLT effects in a cuprizone-induced demyelination model. The

results show that LLLT-treated mice had an improvement in motor coordination, an increase in OPCs

proliferation, and a modulation of astrocytes, microglia and tissue toxicity.

Although cuprizone action mechanisms are still obscure, a non-related food intake/weight loss

phenotype is described in most rodent-cuprizone experiments [35, 36]., as also observed in CPZ and

CPZL animals in this study that exhibited similar chow intake compared to CTL group. It is believed that

weight changes occur in association with sickness behaviour because of cuprizone intoxication and

possible metabolic disturbances [9, 36].

In the CNS, cuprizone demyelination particularly affects the corpus callosum, but the striatum,

hippocampus, cerebellum and brain cortex can also be affected [38, 39, 40, 41, 42]. Corpus callosum is

an important myelinated, connective, inter-hemispheric structure, aggregating bundles from

somatosensorial and motor pathways, and its demyelination leads to motor dysfunction [43, 44].

The accelerated rotarod test was used to verify rodents’ motor coordination in a rotating cylinder.

Detection of motor abnormality becomes more sensitive by continually increasing rpm, once the speed

challenges the animal to adjust its gait [45, 46, 47]. As observed in CPZ mice, lower latencies and high

fall numbers indicates motor impairment, probably due to saltatory conduction loss in myelinated fibres

[44]. A better motor performance in CPZL and CTL groups was observed compared to CPZ group,

suggesting that LLLT might act on corpus callosum demyelination by attenuating or reversing clinical

signs.

In traumatic brain injury (TBI) model, using the LLLT dosage of 36J/cm2 energy density and

wavelength of 810 nm results in an increase in Brain-Derived Neurotrophic Factor (BDNF) expression,

enhanced synaptogenesis, decreased pro-inflammatory cytokines expression in brain, reduction in both

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lesion extension and secondary injury of nervous tissue, and improved clinical outcome [48, 49, 50,51,

52].

It is important to highlight that treatment with LLLT in CNS lesion models results in better

functional recovery. In TBI model, improvements have been verified in neurological signs of cognition and

motor function in laser-treated mice [27]. In spinal cord injury models, laser therapy has been associated

with axon regeneration and better functional outcome [ 53, 54, 55].

Lactate Dehydrogenase (LDH) was analysed for cuprizone toxicity as serum levels are elevated

in necrotic lesions, when cytoplasmic membrane integrity is broken, and this enzyme is released into

extracellular space [56]. Previous studies found that the initial phase of cuprizone exposure is marked by

apoptosis, but when the treatment is administrated for more than four weeks, necrosis can be triggered

by lipid peroxidation during oxidative stress, by TNF-α family pro-inflammatory cytokines or by ATP

depletion [57, 58]. CPZ group presented significantly higher levels of LDH compared to CTL group,

probably due to cuprizone-induced demyelination reaching a significant level of cell damage due to

energy depletion and severe inflammation. Interestingly, CPZL group presented no statistically significant

difference to CTL or CPZ groups. It is accepted that a reduction in LDH levels indicates less damage to

the cells. In this study, animals from CPZL group treated with LLLT, consistently exhibited lower levels of

serum LDH, suggesting some positive effect of the laser therapy on the lesion [59]

After four complete weeks of continuous cuprizone diet, severe demyelination takes place in

mice’ corpus callosum, as is well documented in literature, [11, 60] and this was evident in CPZ group.

The myelin specific LFB dye is an alcoholic copper phthalocyanine solution that, when in contact with

myelin lipids, leads to a simple exchange reaction, resulting in a blue precipitate [61, 62]. In mature

oligodendrocytes, over 25-30% of myelin composition corresponds to MBP, an intracellular protein that

compacts myelin by organizing it in a multilamellar structure [29, 64, 65]. In CPZ animals’, corpus

callosum demyelination is shown as areas of pale LFB staining, and reduced MBP immunostaining,

associated with myelin loss due to oligodendrocyte degeneration [66, 67, 68, 69]. Interestingly, LLLT

probably attenuated or reversed the degree of demyelination in the CPZL corpus callosum, as evidenced

by a higher LFB and MBP density than CPZ group. This could be due to a preserved or recovered

saltatory conduction, resulting in better clinical outcome [44, 67].

Studies that verify the effect of LLLT in myelinating cells have been mostly directed to the

peripheral nervous system. In vitro, the treatment stimulated Schwann cells proliferation and upregulated

Nerve Growth Factor (NGF) expression [70]. In vivo, LLLT increased the number of peripheral myelinated

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fibres of sciatic nerve-injured rats and improved clinical outcome [24, 71]. Previous studies suggest that

neuronal survival may be explained in part by mitochondrial function restoration, downregulation of

inflammation, oxidative stress inhibition, and modulation of apoptosis proteins [22,72].

In Experimental Autoimmune Encephalomyelitis (EAE), a reduction of oxidative stress and

upregulation of anti-apoptotic proteins expression in spinal cord has been observed [73]. Gonçalves and

cols., applying wavelengths of 660 and 904nm during the first 30 days post-immunization of EAE, showed

significantly milder clinical signs in the animals that received transcranial low-level laser therapy (TLLLT)

and these results were reinforced by the reduction in the neuroinflammatory process. Additionally,

demyelination, inflammatory cytokine levels and nitric oxide were modulated [74].

It is generally considered that experimental models of multiple sclerosis fulfilled all the clinical

signs and mechanisms of this disease. In fact, each experimental model allowed the analysis of specific

aspects of MS. As suggested by Franklin and ffrench-Constant [75] in a recent review, EAE is a model of

the immunopathogenesis of MS, which mimics the inflammatory process.

In this study, the cuprizone was used as a model of MS to investigate the effect of TLLLT on

demyelination. Although the cuprizone action mechanism is not completely understood, it is known that it

induces mitochondrial dysfunction, possibly affecting cytochrome c oxidase activity, resulting in

mitochondrial damage, oxidative stress and energy failure [76, 77]. Indeed, oligodendrocytes produce and

maintain myelin sheaths in CNS, with high energy consumption, and it is believed that cuprizone leads to

oligodendrocyte apoptosis through damage to respiratory chain in mitochondria [13, 78]. The

neuroprotector role of infrared low-level laser seems to act on mitochondria by exciting cytochrome c

oxidase, intensifying the respiratory chain activity acting on the cytochrome c oxidase activity and, in

consequence, ATP production [79,80]. Besides, it has been shown that transcranial laser therapy also

preserves mitochondria improving its antioxidant capability [80].

Associating the considerations that cuprizone might lead to demyelination by impairing

mitochondrial function, and that LLLT may contribute to mitochondrial function, it is likely that this therapy

protects oligodendrocytes from degeneration or stimulates oligodendrogenesis.

The transcription factor Olig2 characterizes oligodendrocyte lineage cells, and controls OPCs’

migration, differentiation and oligodendrocyte maturation [81,82]. Although cuprizone induces

oligodendrocyte death, a depletion of Olig2 positive cells would be expected in corpus callosum.

However, in the 1970s, OPCs were found during severe demyelination phase of cuprizone [83].

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Subsequent studies revealed that cuprizone is not toxic for OPCs and does not affect OPCs’ proliferation,

although it inhibits the differentiation [15]. As observed in this study during quantification of CPZ and

CPZL Olig2 positive cells, and noted by Mason et al., the number of Olig2 positive cells is unchanged or

increased in the fourth week of cuprizone, probably due to OPCs’ recruitment within corpus callosum [84].

The significantly higher number of these cells in CPZL suggests a stronger recruitment of OPCs and

therefore, their proliferation quantification was verified by co-localization of PDGFβR/Ki67.

In immunohistochemistry, it is possible to use PDGFβR as a marker for OPCs [85, 86],

associated with Ki67, a nuclear protein expressed during cell proliferation [87], to identify the proliferating

OPCs. After lesion, OPCs’ proliferation increases, mainly in response to the inflammatory clues like

cytokines [88].

OPC dynamics is influenced by growth factors expressed during neuroinflammation, such as

Fibroblast Growth Factor-1 (FGF-1), Brain-Derived Neurotrophic Factor (BDNF), PDGF, and Insulin-like

Growth Factor-1 (IGF-1) [89, 90, 91, 92]. Since CTL group was not demyelinated by cuprizone, very few

OPCs in proliferation were seen in the corpus callosum. Conversely, there was a higher number of these

cells in CPZ group because cuprizone exposure leads to OPCs’ recruitment and proliferation during

severe phase of demyelination [83, 93]. Xuan and cols. observed that LLLT stimulated neural precursor

cells, associated with neurogenesis, in traumatic brain injury (TBI) model. Indeed, our results also

suggest that LLLT enhanced OPC proliferation, as a higher number of OPCs was found in the CPZL

corpus callosum [53].

An efficient remyelination depends on debris clearance and modulation of neuroinflammation by

microglia and astrocytes [94]. Particularly, adult myelin debris contains anti-regenerative proteins, such as

Myelin-Associated Glycoprotein (MAG), Myelin Oligodendrocyte Glycoprotein (MOG), and Nogo-A, which

inhibit OPCs differentiation. In demyelinating diseases, with each oligodendrocyte degeneration cycle,

myelin debris accumulates in the lesion, impairing remyelination [95,96]. Microglia/macrophages and

astrocytes are paradoxical cells because these inflammatory cells are indispensable for remyelination,

expressing growth factors that will provide conditions for the regeneration [6, 94].

Cuprizone affects oligodendrocytes mainly during early demyelination [76]. From the second

week of cuprizone use, myelin debris activates and attracts microglia within demyelination sites, which is

followed, in the third week, by infiltration of macrophages plus astrocytes activation and proliferation. Pro-

inflammatory cytokines, expressed by activated microglia/macrophages and astrocytes, promote

neuroinflammation and death of secondary oligodendrocytes [11, 97, 98]. When activated, microglia

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swell, increase cell perimeter, and retract cytoplasmic processes, changing to a macrophage-like

morphology [99]. Demyelination triggers activation of astrocytes, which proliferate and expand

cytoplasmic processes, increasing their perimeter [98, 100]. Studies of the modulation of microglia and

astrocytes by low-level laser irradiation are still in the early stages, and there are few studies elucidating

its mechanisms.

Nevertheless, the complete eradication of the activation of microglia and astrocytes could impair

regeneration. It has been shown that the absence of microglia resulted in failed OPC recruitment and

differentiation, probably due to the presence of myelin debris and reduced release of growth factors [101].

Skripuletz et al. have demonstrated that ablation of astrocytes also impairs remyelination, in which myelin

clearance by microglia is affected. This suggests that astrocytes control microglial phagocytosis, which is

important for the removal of myelin inhibitory proteins within debris [102].

In this work, microglia and infiltrated macrophages (M/M) were identified by IBA-1

immunostaining. IBA-1 is a cytosolic protein that interacts with actin microfilaments by reorganizing

cellular shape when microglia become activated, resulting in upregulation of IBA-1 expression and

increased immunostaining [103,104]. It has been shown that microglia culture irradiated with low-level

laser downregulated both Tumour Necrosis Factor-Alpha (TNF-α) and inducible nitric oxide synthase

(iNOS) expression, and improved the phagocytic function [32]. It has also been demonstrated that LLLT

modulates microglia activation in a TBI model [105]. The corpus callosum in the CPZL group showed

reduced intensity IBA-1 immunostaining, with lower quantity of IBA-1 positive cells with smaller

perimeters compared to CPZ group. It was notable that although most cells in CPZL group had

macrophage-like morphology, some of them presented reduced perimeters. All these findings taken

together, suggest that LLLT modulated microglia activation. Consequently, it is quite feasible that this

modulation, implied by debris clearance improvement and reduced neuroinflammation, could allow

demyelination attenuation and/or remyelination.

Microglia and macrophages can function in two known states: the M1 or M2 phenotypes.

Function of M1 polarization is related to a neurotoxic and pro-inflammatory role, whereas M2 is

associated with anti-inflammatory and reparative status. It is believed that increasing M2 polarization

and/or reducing M1 phenotype may promote neural tissue recovery. A recent study showed that LLLT

altered the polarization of microglia, leading to an M2 propensity. This was correlated with boosted

neuroprotection and a shortened lesion area [106].

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In the current study, astrocytes were analysed by GFAP immunostaining an intermediate

filament. During acute demyelination induced by cuprizone, activated astrocytes enhance inflammation

microenvironment and release fibronectin aggregates, a potent OPC differentiation inhibitor [14, 107].

Yang et al. have observed that amyloid-beta peptide-induced activated astrocytes in vitro were modulated

by LLLT, evidenced by suppression of the pro-inflammatory Interleukin-1 beta and iNOS [31]. A model of

Parkinson disease demonstrated that infrared laser was effective in downregulating astrogliosis [107]. In

the present study, the reduced number and perimeter of GFAP positive cells in the CPZL corpus

callosum, compared to CPZ group, probably indicates that LLLT modulated astrogliosis, without

extinguishing astrocytes activation [108]. In a recent study, Yang and cols. showed in a model of ischemic

stroke, a modulation of inflammatory process promoted by photobiomodulation, inducing the microglia M2

phenotype and astrocytes activation [109].

Remyelination is a fundamental step for saltatory conduction restoration. At the same time, it

remains blockaded by molecular and inflammatory barriers [110]. Strategies for dealing with remyelination

and inflammatory modulation are essential for axonal preservation and functional outcome recovery of

patients with demyelinating diseases [111]. However, it will be necessary to adjust laser parameters for

human patients since the skull thickness is substantially greater [112]. In studies using cadaver human

heads, an 808 nm wavelength of low-level laser showed the best penetration of the skull/brain [112,113].

Laser treatment does not produce adverse effects in normal tissue. Past studies confirm that

enzymes, nervous function, myelin density, neurite outgrowth, and apoptosis rates were not altered in

normal rodents submitted to LLLT [25,69,114,115,116]. In another study, a monkey’s brain was implanted

with an optical fibre delivering red light to evaluate long-term effects on normal brain tissue. No signs of

toxicity, histological abnormalities nor activating glial response signs were noted [117].

Our results suggest there are beneficial effects of LLLT on motor coordination, demyelination

attenuation, OPCs proliferation stimuli, and modulation of microglia and astrocytes.

Conclusion

Low-level laser therapy (LLLT) has been shown to be advantageous in motor coordination

recovery in demyelinated mice. This may in part, be associated with attenuated demyelination and/or

proliferation of OPCs. Milder oligodendrocyte damage can be linked to modulation of neuroinflammation

supported by microglia/macrophages and astrocytes reduced number induced by laser.

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Neuroinflammation modulation may have a protective role for OPCs proliferation, and thereafter, is

important for oligodendrocytes differentiation. Collectively, the results of this work suggest that low-level

laser presents a promising therapy for remyelination.

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Acknowledgements

The authors would like to say thank you to Tony Champion for his carefully English review and

suggestions.

Funding

This work was funded by the National Council for Scientific and Technological Development (CNPq),

Coordination for the Improvement of Higher Education Personnel (CAPES and São Paulo Research

Foundation, São Paulo, Brazil (FAPESP- 2007/07828-3).

Conflict of Interest

The authors declare that they have no conflict of interest.

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Figure 1. Assessment of motor coordination by rotarod test. The rotarod test can be used as a

functional test, since demyelination can result in a range of clinical signs as a result of myelin

degeneration blocking nerve impulses. (a) Latency to fall - Mice from CTL group exhibited the longest

latency to fall, expected for normal motor coordination, i.e., animals were able to re-adjust their gait as the

rotation speed of the cylinder accelerated. In CPZ group animals, a statically significant shorter latency to

fall was observed, especially at the highest speed. As a suggestion of recovery, CPZL group showed a

latency to fall close to the CTL group. (Diagram (a): mean values ± S.E.M.; ∗ p=0.0201; F=5.510). (b)

Number of falls – In line with the latency to fall results, on the 28th day of the experiment, CPZ group

exhibited a number of falls significantly higher in relation to CTL group, whereas CPZL mice

demonstrated a similar performance to CTL group (Diagram (b): mean values ± S.E.M.; ∗ p<0.05;

H=5.846).

Figure 2. Lactate dehydrogenase assay. Serum LDH is a tissue damage marker. As expected, the CTL

group exhibited the lowest levels of LDH. Exposure to cuprizone significantly increased these levels. As

can be observed in CPZ group, the LDH concentration was significantly higher compared to CTL, while

CPZL serum exhibited an intermediate concentration, neither different from CTL nor CPZ serum

concentration (mean values ± S.E.M.; p<0.05; H=6.25).

Figure 3. Myelin semi-quantification analysis. LFB is a stain specific to lipids, in a normal white matter

appearing as a deep blue colour. CTL group exhibited dense and uniform (LFB staining in corpus

callosum (a). CPZ group showed the lightest staining and tissue frangibility because of demyelination (b).

In CPZL group, it was possible to observe the effect of the LLLT on demyelination: the staining is darker

and the brain tissue displays a well preserved structure (c). Semi-quantification analysis showed density

of myelin significantly lower in CPZ group in comparison to CTL group, but no difference was observed

between CPZL and CTL groups ((g); mean values ± S.E.M.; ∗ p=0.0275; H=7.19). These results

reinforce the hypothesis that there is a potentially therapeutic effect of 808nm laser on myelin. MBP, a

structural protein expressed in mature oligodendrocytes, is important for multilamellar myelin sheath

structure. (d) In the CTL corpus callosum group MBP immunostaining characteristic of the intact myelin,

while a significantly lower immunolabelling was seen in CPZ group (e) and CPZL corpus callosum group

showed a significantly milder myelin loss compared to CPZ group, a fact that suggests an attenuation of

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demyelination or remyelination stimulated by low-level laser as demonstrated by significantly reduced

fluorescence intensity (h; mean values ± S.E.M.; ∗ p<0.05; ∗ ∗ p<0.001; ∗ ∗ ∗ p<0.0001; H=38.11).

LFB: luxol fast blue; MBP: myelin basic protein.

Figure 4. Oligodendrocyte lineage cells immunostaining analysis in the corpus callosum.

Olig2 is a transcription factor expressed by all oligodendrocyte lineage cells. As a rich myelinated

structure, most of the Olig2+ cells present in corpus callosum are mature oligodendrocytes, as seen in

CTL group (a). Due to demyelination, Olig2+ cells lost their organization and OPCs were recruited, as can

be seen in CPL (b) and CPLZ groups (c). Demyelination induces the proliferation and migration of OPCs,

as reflected in the quantity of these cells compared to healthy animals, visible in CPZL group (d - mean

values ± S.E.M.; ∗ ∗ p<0.001; H= 9.529). Olig2: Oligodendrocyte transcription factor; OPC:

Oligodendrocyte precursor cells.

Figure 5 Co-localization PDGF-Rβ and Ki67 positive cells in the corpus callosum.

PDGF-βR is expressed in cell membranes of OPCs and Ki67 is a protein regulator of cell proliferation.

The co-localization of PDGF-Rβ and Ki67 proteins were used to analyse OPCs proliferating and migrating

to demyelinated lesion. In the adult CNS, only a few cells are in proliferation, as observed in CTL group

(a). The cells are activated in response to growth factors released by inflammatory cells and astrocytes

during the lesion, leading to proliferation and migration to corpus callosum, as seen in CPZ group (b). In

the CPZL group (c), the proliferation of OPCs was higher compared to CPZ group, which can be

associated with an enhancing proliferation effect of LLLT (d; mean values ± S.E.M.; p<0.001;

p<0.0001; H=42.04). PDGF-Rβ: platelet-derived growth factor receptor beta.

Figure 6. Microglia and astrocytes immunohistochemistry. Microglia and astrocytes are the main cell

types to handle the inflammatory process in CNS. After lesion, those cells become activated and change

their morphology. (a) Using Immunolabelling to IBA1, in CTL corpus callosum it was possible to observe

mainly quiescent microglia, exhibiting ramified branches and small cells. (b and c) As treatment with

cuprizone induces demyelination, these cells change their shape assuming a macrophage-like

morphology as shown in CPZ and CPZL groups. Semi-quantitative analyses show a significantly higher

quantity and perimeter of IBA+ cells in CPZ group, when compared with CTL and CPZL (d and e). These

features are reflected in a significantly higher intensity of IBA-1+ cells in CPZ group in relation to CTL and

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CPZL (f; H= 44.59 for quantity, 29.67 for perimeter, 37.05 for intensity). Astrocytes in healthy nervous

tissue exhibit fine-branched GFAP+ cells, as observed in CTL corpus callosum (g). After lesion, the

expression of GFAP and the cell branches is increased, resulting in intense immunostaining, as shown in

CPZ group (h) and, to a lesser extent, in CPZL group (i). Cell counts showed CPZ group mice had a

significantly higher number of GFAP+ cells in comparison with CTL and CPZL groups (j). CTL group

presented a smaller diameter (k) in comparison with CPZ and CPZL. Consistent with previous results, the

fluorescence intensity was also higher in CPZ group in comparison to CTL and CPZL (l; mean values ±

S.E.M.; *p<0.05; p<0.001; p<0.0001; H=40.72 for quantity, 72.73 for perimeter, 42.83 for intensity).

IBA1: Ionized calcium binding adaptor molecule 1; GFAP: glial fibrillary acidic protein.

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Highlights

LLLT improved motor coordination in demyelination induced by cuprizone in mice

Demyelinated animals treated with laser showed oligodendrocytes lineage proliferation

LLLT can modulate microglia and astrocytes activation in cuprizone model

Laser therapy appears to mitigate demyelination induced by cuprizone

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