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Low-level Laser (Light) Therapy Increases Mitochondrial Membrane Potential and ATP Synthesis in C2C12 Myotubes with a Peak Response at 3–6 h


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Low level laser (light) therapy has been used before exercise to increase muscle performance in both experimental animals and in humans. However uncertainty exists concerning the optimum time to apply the light before exercise. The mechanism of action is thought to be stimulation of mitochondrial respiration in muscles, and to increase adenosine triphosphate (ATP) needed to perform exercise. The goal of this study was to investigate the time course of the increases in mitochondrial membrane potential (MMP) and ATP in myotubes formed from C2C12 mouse muscle cells and exposed to light-emitting diode therapy (LEDT). LEDT employed a cluster of LEDs with 20 red (630 ± 10 nm, 25 mW) and 20 near-infrared (850 ± 10 nm, 50 mW) delivering 28 mW/cm(2) for 90 sec (2.5 J/cm(2) ) with analysis at 5 min, 3 h, 6 h and 24 h post-LEDT. LEDT-6h had the highest MMP, followed by LEDT-3h, LEDT-24h, LEDT-5min and Control with significant differences. The same order (6h>3h>24h>5min>Control) was found for ATP with significant differences. A good correlation was found (r=0.89) between MMP and ATP. These data suggest an optimum time window of 3-6 h for LEDT stimulate muscle cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Photochemistry and Photobiology, 20**, **: **
Low-level Laser (Light) Therapy Increases Mitochondrial Membrane
Potential and ATP Synthesis in C2C12 Myotubes with a Peak Response
at 36h
Cleber Ferraresi
, Beatriz Kaippert
, Pinar Avci
, Ying-Ying Huang
, Marcelo V. P. de Sousa
Vanderlei S. Bagnato
, Nivaldo A. Parizotto
and Michael R. Hamblin
Laboratory of Electrothermophototherapy, Department of Physical Therapy, Federal University of Sao Carlos, Sao Carlos,
SP, Brazil
Post-Graduation Program in Biotechnology, Federal University of Sao Carlos, Sao Carlos, SP, Brazil
Optics Group, Physics Institute of Sao Carlos, University of S~
ao Paulo, Sao Carlos, SP, Brazil
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA
Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Department of Dermatology, Harvard Medical School, Boston, MA
Laboratory of Radiation Dosimetry and Medical Physics, Institute of Physics, Sao Paulo University, Sao Carlos, SP, Brazil
Harvard-MIT Division of Health Science and Technology, Cambridge, MA
Received 2 September 2014, accepted 25 November 2014, DOI: 10.1111/php.12397
Low-level laser (light) therapy has been used before exercise
to increase muscle performance in both experimental animals
and in humans. However, uncertainty exists concerning the
optimum time to apply the light before exercise. The mecha-
nism of action is thought to be stimulation of mitochondrial
respiration in muscles, and to increase adenosine triphosphate
(ATP) needed to perform exercise. The goal of this study was
to investigate the time course of the increases in mitochondrial
membrane potential (MMP) and ATP in myotubes formed
from C2C12 mouse muscle cells and exposed to light-emitting
diode therapy (LEDT). LEDT employed a cluster of LEDs
with 20 red (630 10 nm, 25 mW) and 20 near-infrared
(850 10 nm, 50 mW) delivering 28 mW cm
for 90 s
(2.5 J cm
) with analysis at 5 min, 3 h, 6 h and 24 h post-
LEDT. LEDT-6 h had the highest MMP, followed by LEDT-
3 h, LEDT-24 h, LEDT-5 min and Control with signicant
differences. The same order (6 h >3h>24 h >5 min >
Control) was found for ATP with signicant differences. A
good correlation was found (r=0.89) between MMP and
ATP. These data suggest an optimum time window of 36h
for LEDT stimulate muscle cells.
Mitochondria are the organelles responsible for energy produc-
tion in cells and for this reason have a very important role in cel-
lular function and maintenance of homeostasis. This organelle
has an intriguing and well-designed architecture to generate
adenosine triphosphate (ATP) that is the basic energy supply for
all cellular activity (1,2).
Mitochondria contain a respiratory electron transport chain
(ETC.) able to transfer electrons through complexes I, II, III and
IV by carrying out various redox reactions in conjunction with
pumping hydrogen ions (H
) from the mitochondrial matrix to
the intermembrane space. These processes generate water as the
metabolic end-product, as oxygen is the nal acceptor of elec-
trons from the ETC., that is coupled with synthesis of ATP when
ions return back into mitochondrial matrix through complex
V (ATP synthase), thus completing the ETC. Changes in the
ow of electrons through the ETC. and consequently in H
pumping produce signicant modulations in the total proton
motive force and ATP synthesis. These changes can be measured
by mitochondrial membrane potential (MMP) and content of
ATP (1).
Since the earliest evidence that low-level laser (light) therapy
(LLLT) can increase ATP synthesis (3,4), several mechanisms of
action have been proposed to explain LLLT effects on mitochon-
dria. One of the rst studies reported increased MMP and ATP
synthesis measured at an interval of 3 min after LLLT (3). Years
later, other authors extended the measurement of this extra
ATP-induced by LLLT in HeLa (human cervical cancer) cells
(4). With intervals of 5 to 45 min, these authors found no
change in ATP synthesis during the rst 15 min after LLLT, but
after 2025 min ATP levels increased sharply and then came
back to control levels at 45 min (4).
More recent studies have reported LLLT effects on mitochon-
dria in different types of cells (59). In neural cells LLLT seems
to also increase MMP, protect against oxidative stress (5) and
increase ATP synthesis in intact cells (without stressor agents)
(6). In mitochondria from broblast cells without stressor agents,
LLLT also increased ATP synthesis and mitochondrial complex
IV activity in a dose-dependent manner (7). In myotubes from
C2C12 cells, LLLT could modulate the production of reactive
oxygen species (ROS) and mitochondrial function in a dose-
dependent manner in intact cells or in cells stressed by electrical
stimulation (9).
Increases in mitochondrial metabolism and ATP synthesis
have been proposed by several authors as a hypothesis to explain
*Corresponding author email: (Michael R.
©2014 The American Society of Photobiology
LLLT effects on muscle performance when used for muscular
preconditioning or muscle recovery postexercise (1012). How-
ever, there is a lack in the literature to identify immediate and
long-term effects of LLLT on mitochondrial metabolism and
ATP synthesis in skeletal muscle cells that in turn could conrm
these hypotheses.
This study aimed to identify the time-response for LLLT by
light-emitting diode therapy (LEDT) in modulation of MMP and
ATP content in myotubes from C2C12 intact cells (mouse mus-
cle cells) only under the stress of the culture. Moreover, the sec-
ond objective was to correlate MMP with ATP content within a
time range of 5 min to 24 h after LLLT. Our goal was to nd
the best time-response for LLLT which could be useful in future
experimental and clinical studies investigating muscular precon-
ditioning, muscle recovery postexercise or any other photobio-
modulation in muscle tissue.
Cell culture. C2C12 cells were kindly provided by the Cardiovascular
Division of the Beth Israel Deaconess Medical Center, Harvard Medical
School, USA. Cells were grown in culture medium (DMEM, Dulbeccos
Modied Eagles Medium - Sigma-Aldrich) with fetal bovine serum
(20% FBS - Sigma-Aldrich) and 1% antibiotic (penicillin and streptomy-
cin) in humidied incubator at 37°C and 5% CO
C2C12 cells were cultured and a total of 1.71 910
cells approxi-
mately were counted in a Neubauer chamber. Next, these cells were dis-
tributed equally into 30 wells (approximately 5.7 910
cells per well)
into two different plates:
1 15 wells in black plate (Costar
96-Well Black Clear-Bottom Plates)
for analysis of MMP.
2 15 wells in white plate (Costar
96-Well White Clear-Bottom Plates)
for analysis of ATP synthesis.
Moreover, both plates were subdivided into ve columns with three
wells per column (triplicate):
1 LEDT-Control: no LEDT applied to the cells.
2 LEDT-5 min: LEDT applied to the cells and assessments of ATP and
MMP after 5 min.
3 LEDT-3 h: LEDT applied to the cells and assessments of ATP and
MMP after 3 h.
4 LEDT-6 h: LEDT applied to the cells and assessments of ATP and
MMP after 6 h.
5 LEDT-24 h: LEDT applied to the cells and assessments of ATP and
MMP after 24 h.
After plating C2C12 cells were cultured for 9 days in culture medium
(DMEM) containing 2% heat-inactivated horse serum (Sigma-Aldrich) in
a humidied incubator at 37°C and 5% CO
to induce cell differentiation
into myotubes, as described in a previous study (9). At the 10
LEDT-24 h group received LEDT. At 11
day all remaining groups
received LEDT and were assessed for ATP and MMP at specic times in
accordance with each group.
Light-emitting diode therapy (LEDT). A cluster of 40 LEDs (20 red
630 10 nm; 20 infrared 850 20 nm) with a diameter of 76 mm
was used in this study. The cluster was positioned at a distance of
156 mm from the top of each plate and irradiation lasted 90 s with xed
parameters as described in Table 1. Each group of wells received LEDT
individually, and all others wells of each plate (groups) were covered
with aluminum foil to avoid light irradiation (Fig. 1). LEDT parameters
were measured and calibrated using an optical energy meter PM100D
and sensor S142C (area of 1.13 cm
). In addition, we chose
use red and near-infrared light therapy at the same time to promote a
double band of absorption by cytochrome c oxidase (Cox) based on spe-
cic bands of absorption reported previously (2,1316). The room tem-
perature was controlled (2223°C) during LEDT irradiation, which did
not increase temperature on the top of plates more than 0.5°C. This
increase of 0.5°C was dissipated to room within 2 min after LEDT.
Mitochondrial membrane potential (TMRM) assay. This analysis was
performed using cells placed into black plate. MMP was assessed using
tetramethyl rhodamine methyl ester (TMRM Invitrogen/Molecular
Probes) at a nal concentration of 25 nM. Nuclei of myotubes from
C2C12 cells were labeled using Hoechst (Sigma-Aldrich) at a concentra-
tion of 1 mg mL
. Each well was incubated for 30 min, 37°C and 5%
with 100 lL of solution containing TMRM and Hoechst. Next, this
solution was carefully removed from each well and added 100 lL of buf-
fer solution containing HBSS (Hanks Balanced Salt Solution Life
Technologies Corporation) and 15 mM HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid Life Technologies Corporation). The
myotubes were imaged in a confocal microscope (Olympus America Inc.
Center Valley, PA) using an excitation at 559 nm and emission at
610 nm. Three random elds per well were imaged with a magnication
of 409water immersion lens. Images were exported and TMRM uores-
cence incorporation into mitochondrial matrix was measured using soft-
ware Image J (NIH, Bethesda, MD).
Adenosine triphosphate (ATP) assay. This analysis was performed
using cells placed into white plate. First, the medium was carefully
removed from each well followed by addition of 50 lL per well of
CellTiter Glo Luminescent Cell Viability Assay reagent (Promega). After
10 min of incubation at room temperature (25°C), luminescence signals
were measured in a SpectraMax M5 Multi-Mode Microplate Reader
(Molecular Devices, Sunnyvale, CA) with integration time of 5 s to
increase low signals (17). A standard curve was prepared using ATP
standard (Sigma) according to manufacturers guideline and then ATP
concentration was calculated in nanomol (nmol) per well.
Pearson product-moment correlation coefcient (Pearsons r). The
correlation between TMRM and ATP content in myotubes from C2C12
cells was calculated using Pearsons r. The rvalues were interpreted as
recommended previously (18): 0.000.19 =none to slight; 0.200.39 =
low; 0.400.69 =modest; 0.700.89 =high; and 0.901.00 =very high.
Sample size calculation. The sample size was calculated based on that
necessary to obtain signicant differences among all groups with ATP
Table 1. All parameters of light-emitting diode therapy (LEDT). Control
did not receive LEDT.
Number of LEDs (cluster): 40 (20 infrared-IR and 20 red-RED)
Wavelength: 850 20 nm (IR) and 630 10 nm (RED)
LED spot size: 0.2 cm
Pulse frequency: continuous
Optical output of each LED: 50 mW (IR) and 25 mW (RED)
Optical output (cluster): 1000 mW (IR) and 500 mW (RED)
LED cluster size: 45 cm
Power density (at the top of plate): 28 mW cm
Treatment time: 90 s
Cluster energy density applied on the top plate: 2.5 J cm
Application mode: without contact
Distance from plate or power meter: 156 mm
Figure 1. Myotubes from C2C12 cells. Experimental setup for irradia-
tion of the white and black plates containing myotubes from C2C12 cells
using light-emitting diode therapy (LEDT) without contact.
2 C. Ferraresi et al.
content. The statistical power of 80% and the effect size (greater than
0.75) were found to be satisfactory.
Statistical analysis. ShapiroWilks W test veried the normality of
the data distribution. ATP and TMRM were compared among all groups
using one-way analysis of variance (ANOVA) with Tukey HSD post hoc
test. Pearson product-moment correlation coefcient (Pearsons r) was
conducted between TMRM and ATP. Signicance was set at P<0.05.
Mitochondrial membrane potential (TMRM)
LEDT-6 h group increased MMP (10.77 AU, SEM 0.88) com-
pared to: Control (3.79 AU, SEM 0.46): P<0.001; LEDT-5 min
(4.11 AU, SEM 0.52): P<0.001; LEDT-24 h (4.91 AU, SEM
0.47): P=0.001. LEDT-3 h (7.87 AU, SEM 0.59) increased
MMP compared to Control (P=0.019) and LEDT-5 min
(P=0.031). These results are graphically presented in Fig. 2. All
nonsignicant results were Control versus LEDT-5 min
(P=0.997) and versus LEDT-24 h (P=0.816); LEDT-5 min
versus LEDT-24 h (P=0.935); LEDT-3 h versus LEDT-6 h
(P=0.113) and versus LEDT-24 h (P=0.103).
ATP assay
LEDT-6 h increased ATP contents (4.53 nmol per well, SEM
0.19) compared to: Control (1.28 nmol per well, SEM 0.05):
P<0.001; LEDT-5 min (2.01 nmol per well, SEM 0.16):
P<0.001; LEDT-24 h (2.77 nmol per well, SEM 0.16): P=
0.007. LEDT-3 h increased ATP contents (3.73 nmol per well,
SEM 0.17) compared to Control (P<0.001) and LEDT-5 min
(P=0.008). LEDT-24 h increased ATP contents compared to
Control (P=0.020). These results are graphically presented in
Fig. 3A. All nonsignicant results were Control versus LEDT-
5 min (P=0.385); LEDT-3 h versus LEDT-6 h (P=0.299)
and versus LEDT-24 h (P=0.169); LEDT-24 h versus LEDT-
5 min (P=0.338).
Sample size
The statistical power and the effect size regarding ATP content
in all groups were calculated to ensure the minimal power of
80% and large effect size (>0.75). We used the mean ATP con-
tent of each group and the highest value of standard deviation
among all groups, which was observed in LEDT-6 h. Our results
demonstrate a difference between groups with a statistical power
of 99%, effect size of 3.42 (very large effect) and total sample
size of 10, i.e. 2 wells per group (ve groups). These calcula-
tions demonstrate that our sample size was small, but adequate
(3 wells per group).
Pearson product-moment correlation coefcient (Pearsonsr)
TMRM incorporation into mitochondrial matrix of myotubes
from C2C12 cells showed a high correlation (r=0.89) with
ATP content (P<0.001). This result is presented in Fig. 3B.
This study identied a well-dened time-response for the LEDT-
mediated increase in MMP and ATP synthesis in myotubes from
C2C12 cells under the stress of the cell culture. The light dose
used was based on previous study that already reported benets
of LLLT on mitochondria of myotubes (9). In addition, we found
a strong correlation between MMP and ATP content measured
Figure 2. TMRM. Analysis of mitochondrial membrane potential using tetramethyl rhodamine methyl ester (TMRM) stained in red. Images with a
magnication of 409. Abbreviations: LEDT=light-emitting diode therapy; AU =arbitrary units; C =control group; 5 min =LEDT-5 min group;
24 h =LEDT-24 h group; *=statistical signicance (P<0.05) using one-way analysis of variance (ANOVA).
Photochemistry and Photobiology 3
during a wide range from 5 min (immediate effect) to 24 h
(prolonged effect). To our knowledge this is the rst study inves-
tigating the time-response for light therapy modulation of mito-
chondrial metabolism in conjunction with ATP synthesis in
muscle cells.
C2C12 is a cell line originally isolated from dystrophic mus-
cles of C3H mice by Yaffe and Saxel (19). In culture it rapidly
differentiates into contractile myotubes (muscle bers) especially
when treated with horse serum instead of fetal bovine serum.
These myotubes contain multinucleated cells that express pro-
teins characteristic of skeletal muscle such as myosin heavy
chain and creatine kinase (20).
One of rst effects of LLLT reported in literature was a mod-
ulation on MMP and ATP synthesis in mitochondria isolated
from rat liver (3) and in HeLa cells (4). Our results are in accor-
dance with these previous studies, showing an increased MMP
and ATP synthesis in myotubes from C2C12 cells. However,
light therapy seems to produce a different time-response of
MMP and ATP synthesis among different cell types. While HeLa
cells showed a peak of ATP synthesis around 20 min after light
therapy (4), mitochondria from liver showed an immediate
increase in MMP and ATP synthesis (3). In this study, we found
that muscle cells need a longer time in the range of 3 h to 6 h to
show the maximum effect of light therapy and convert it into a
signicant increase in MMP and ATP synthesis, comprising an
increase around 200% to 350% over the control values. In addi-
tion, we found that 24 h after irradiation, myotubes could still
produce signicantly more ATP compared to LEDT-Control
while LEDT-5 min showed no signicant difference.
Cytochrome c oxidase (Cox) has been reported to be the main
chromophore in cells exposed to red and near-infrared light
(2,15,16,21). However, although Cox activity is important in the
immediate effects of photon absorption, the measurement of its
activity may be insufcient to conrm whether light therapy can
induce extraATP synthesis. For this reason, the measurement
of MMP in conjunction with ATP synthesis can provide informa-
tion on how fast changes occur in the electron transport chain
(ETC.), and H
pumping from the mitochondrial matrix to the
intermembrane space, as well as how much H
ions are returning
to the mitochondrial matrix (1). In this perspective, our results
are consistent with Xu et al. (9) who reported no immediate
effects of light therapy on MMP. Moreover, although Xu et al.
Figure 3. ATP and Pearsonsr. (A) Analysis of adenosine triphosphate (ATP) content between groups. (B) Pearson product-moment correlation coef-
cient (Pearsonsr) between ATP and mitochondrial membrane potential using TMRM. Abbreviations: LEDT =light-emitting diode therapy;
TMRM =tetramethyl rhodamine methyl ester; nmol =nanomol; AU =arbitrary units; C =control group; 5 min =LEDT-5 min group; 24 h =LEDT-
24 h group; *=statistical signicance (P<0.05) using one-way analysis of variance (ANOVA).
Figure 4. Mechanism of action of LEDT on mitochondria. (A) Mito-
chondria of myotubes from C2C12 cells without low-level laser therapy
(LLLT) or light-emitting diode therapy (LEDT). There is a normal ux
of electrons (red arrow) through all complexes of electron transport chain,
normal pumping of H
, normal synthesis of ATP and modest take up of
TMRM by the mitochondrial matrix. (B) Mitochondria of myotubes from
C2C12 cells 36 h after LEDT. There is an increased ux of electrons
(ticker red arrow), increased pumping of H
, increased synthesis of ATP
and increased take up of TMRM by the mitochondrial matrix. Abbrevia-
tions: I, II, III, IV and V =complexes of the mitochondrial electron
transport chain; H
=proton of hydrogen; - =electron of hydrogen;
=oxygen; H
O=metabolic water; Q =quinone; Cox =cytochrome
c oxidase; ATP =adenosine triphosphate;TMRM =tetramethyl rhoda-
mine methyl ester.
4 C. Ferraresi et al.
(9) did not assess ATP content, our results showed no signicant
responses for ATP increment immediately after light therapy
compared to control group.
Our results for MMP in conjunction with ATP content had a
high correlation (Pearsonsr=0.89) during the time range of
5 min to 24 h, suggesting a linear and positive dependence of
ATP synthesis on the value of MMP (ETC. and H
pumping) in
muscle cells, suggesting a new and more efcient time-response
or time window for LEDT stimulate muscle cells (see Fig. 4A,
B). These results are very important for muscle recovery postex-
ercise (10,11) because they suggest a prolonged effect of light
therapy on ATP synthesis necessary to repair muscle damage. In
addition, muscular preconditioning using light therapy for
improvement of performance before a bout of exercise (12) could
possibly be optimized by application at the appropriate time.
However, more studies in vivo and clinical trials are needed to
conrm our hypotheses.
Muscular preconditioning using LLLT or LEDT have been
reported as therapeutic approaches to improve muscle perfor-
mance in both experimental models (2224) and in clinical trials
(12). However, although this improvement reported in the litera-
ture has been signicant, some studies have not found positive
results (25). Furthermore, differences between groups treated
with light therapy or placebo seem to be not so large. These dif-
ferences reported in experimental models varied between 80%
and 150% of the values found for control groups for fatigue test
induced by electrical stimulation (2224). In clinical trials these
differences varied between 5% and 57% increases in number of
repetitions and maximal voluntary contraction (12). Possibly
these relatively modest increases could be due to allowing insuf-
cient time necessary for the muscle cells to convert light ther-
apy into biological responses as identied in our study for MMP
and ATP synthesis. Consequently, protocols for muscular pre-
conditioning that have been done up to now (12,2224), i.e. gen-
erally applying light 5 min before the exercise, may not possibly
achieve the best result. On the basis of our results, we suggest to
wait 36 h after light therapy irradiation to obtain the best
increase in muscle performance in muscular preconditioning regi-
men, as MMP and ATP availability are important for muscle
performance (26,27). Once more time, we would like to remark
the needed of more studies in vivo and clinical trials to conrm
our hypotheses. At this point, it is valuable to reference two
previous studies that had a similar initiative (28,29). Hayworth
et al. (28) found increments in Cox activity 24 h after apply
LEDT over rats muscles; Albuquerque-Pontes et al. (29) found a
time window, wavelength-dependence and dose response for
Cox activity increase also after LLLT in rats muscles. Both stud-
ies used animals without any kind of stress, such as this study
used cells only under the stress of the cell culture. We believe
that these previous studies combined with our results are extre-
mely valuable for the discovery and understanding of mecha-
nisms of action of LLLT on muscle tissue, and may offer
guidance on the future use of LLLT in clinical practice.
Our study was designed to test one dose of light during a
time-response to show that there is time-dependency for LLLT
to produce secondary responses in muscle cells. For this reason,
this study used a constant dose (uence) of light as reported in a
previous study (9) as well as a constant power density. As there
is a possible biphasic dose response (30,31), use of different
parameters such as uence, wavelengths or irradiance could pro-
duce different responses. In addition, red and near-infrared light
therapy was delivered at the same time to take advantage of the
double bands in Cox to absorb the light (2,1316).
This is the rst study reporting the benets of mixed red and
near-infrared light therapy on MMP in conjunction with ATP
synthesis in myotubes from C2C12 cells (muscle cells from
mice). Moreover, a well-dened time-response was found for the
increase in ATP synthesis mediated by MMP increased by light
therapy in myotubes.
Our data suggest that 36 h could be the best time-response
for light therapy to improve muscle metabolism. In addition, our
results lead us to think there may be possible cumulative effects
if light therapy is applied at intervals less than 24 h that may
have clinical relevance when LLLT is used for muscle postexer-
cise recovery. Finally, we believe that use of light therapy for
muscular preconditioning could be optimized in future studies
whether the time-response for increases in ATP and MMP found
in this study are taken account.
AcknowledgementsWe thank Professor Zoltan Pierre Arany and his
instructor Glenn C. Rowe for the C2C12 cells and Andrea Brissette for
assistance with multiple roles including purchase of reagents. Cleber
Ferraresi thank FAPESP for his PhD scholarships (numbers 2010/07194-
7 and 2012/05919-0). MR Hamblin was supported by US NIH grant
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... Um dos recursos utilizados atualmente dentro da Fonoaudiologia consiste na irradiação de uma fonte de luz 24 . Trata-se da fotobiomodulação, também conhecida como terapia de luz de baixa intensidade, a qual se refere à aplicação de luz vermelha e/ou infravermelha, capaz de induzir o processo fotoquímico, principalmente nas mitocôndrias, e estimular, assim, a produção de trifosfato de adenosina (ATP) 25 . Esta energia luminosa é absorvida pelas células e converte-se em energia química, o que pode estimular a produção de ATP e melhorar o metabolismo celular. ...
... Por apresentar efeitos sobre o sistema muscular, tem sido alvo de estudos por profissionais que atuam na reabilitação muscular. Este recurso ocorre por meio de aplicação de luz monocromática tecidual influenciando a atividade celular, estimulando ou inibindo funções químicas ou fisiológicas 32 , bem como a redução da fadiga muscular 25 , o maior ganho de força 33 e o relaxamento da musculatura de forma mais ágil 34 . Desta forma, o objetivo do presente estudo foi verificar o uso da fotobiomodulação na dificuldade de deglutição em indivíduos que desenvolveram a forma grave da COVID-19. ...
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This study aimed to verify the use of photobiomodulation in swallowing difficulties in individuals who had a severe form of COVID-19. This case report was based on a quasi-experimental, quantitative study, with primary data collected from a non-probabilistic sample of 13 adults (aged ≥ 19 to < 60 years) of both sexes, who had the severe form of COVID-19. Swallowing was assessed with the Dysphagia Risk Assessment Protocol, and the intervention used photobiomodulation. Descriptive statistics were used. It was found that all research participants’ risk for dysphagia in water and pudding swallowing tests improved from before to after the intervention. It can be concluded that photobiomodulation had positive results in speech-language-hearing practice to treat swallowing difficulties in adults who were affected by the severe form of COVID-19, intubated, tracheostomized, and needed an alternative feeding route, as the swallowing difficulties improved. Keywords: COVID-19; Deglutition Disorders; Intubation, Intratracheal; Laser Therapy
... We used a mitochondrial readout related to the effect of light exposure on MMP (ΔΨm) increase [18,19], measured by the MitoTracker fluorescent dye staining. The cell platform was based on BJ human fibroblasts because these cells display a lower MMP heterogeneity than other cell types [7]. ...
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Starting from the discovery of phototherapy in the beginning of the last century, photobiomodulation (PBM) has been defined in late 1960s and, since then, widely described in different in vitro models. Robust evidence indicates that the effect of light exposure on the oxidative state of the cells and on mitochondrial dynamics, suggesting a great therapeutic potential. The translational scale-up of PBM, however, has often given contrasting and confusing results, mainly due to light exposure protocols which fail to adequately control or define factors such as emitting device features, emitted light characteristics, exposure time, cell target, and readouts. In this in vitro study, we describe the effects of a strictly controlled light-emitting diode (LED)-based PBM protocol on human fibroblasts, one of the main cells involved in skin care, regeneration, and repair. We used six emitter probes at different wavelengths (440, 525, 645, 660, 780, and 900 nm) with the same irradiance value of 0.1 mW/cm2, evenly distributed over the entire surface of the cell culture well. The PBM was analyzed by three main readouts: (i) mitochondrial potential (MitoTracker Orange staining), (ii) reactive oxygen species (ROS) production (CellROX staining); and (iii) cell death (nuclear morphology). The assay was also implemented by cell-based high-content screening technology, further increasing the reliability of the data. Different exposure protocols were also tested (one, two, or three subsequent 20 s pulsed exposures at 24 hr intervals), and the 645 nm wavelength and single exposure chosen as the most efficient protocol based on the mitochondrial potential readout, further confirmed by mitochondrial fusion quantification. This protocol was then tested for its potential to prevent H2O2-induced oxidative stress, including modulation of the light wave frequency. Finally, we demonstrated that the controlled PBM induced by the LED light exposure generates a preconditioning stimulation of the mitochondrial potential, which protects the cell from oxidative stress damage.
... The cell and molecular mechanisms of effect of PBM have ended up fairly nicely understood in recent years. Briefly, the stimulatory impact of PBM is primarily based totally on the absorption of blue, green, yellow, red and NIR photons by means of intracellular chromophores placed within the mitochondria, which includes complicated one to complicated five complexes of mitochondria respiration chain, and possibly additionally via way of means of hemoglobin, chromophores with inside the plasma membrane of cells, changing endogenous enzymes and electron transport, thereby growing mitochondrial respiratory and ATP production [54][55][56]. ...
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One of the most important diabetic complications is diabetic neuropathy, which results in impaired wound healing, leading to numerous difficulties and morbidity and mortality, and is the ultimate consequence of micro-and macrovascular disease. It seems. There are several advanced techniques that reverse many degenerative processes and help heal chronic wounds in diabetics. Laser therapy has shown in many studies to improve wound healing in all phases: inflammatory, (2) proliferative, and (3) tissue remodeling. All of the sixteenth subjects were diagnosed with amputation. They all suffer from chronic severe wounds, and they are all diagnosed with diabetic neuropathy. This study used an advanced laser therapy called multi-wavelength interstitial laser therapy. We used 400nm, 450nm, 530nm, 630nm and 808nm. We applied transdermal laser irradiation on a wound area, and beside the wound, we applied interstitial laser irradiation. In interstitial irradiation, the output power at the end of the fiber is 50 mW. In transdermal irradiation the output power was 100mw. The laser was applied continuously with a total energy density of 1 J/cm² at the wound surface and 2 J/cm² in the area adjacent to the wound. All of the laser wavelengths were applied on each point, in both transdermal and interstitial laser irradiation. Patients are treated twice weekly for 6 weeks. Results show a rapid and conclusive effect on wound healing. All of them begin to feel and feel pain. The inflamed sore area is much better. More investigations can be done by regenerative methods combination.
... This increased blood flow and oxygenation may help to enhance cellular metabolism and reduce inflammation, which could potentially lead to lower creatine levels. Additionally, iPBM treatment may also have a direct effect on the mitochondria within cells, leading to improved energy production and metabolism, which could also contribute to reduced creatinine levels [46]. ...
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Background: Although cognitive-behavioral therapy is the first-line treatment for insomnia, pharmacotherapy is often prescribed to treat insomnia and related symptoms. In addition, muscle relaxants are commonly prescribed to alleviate muscle soreness when the pain is unbearable. However, pharmacotherapy can lead to numerous side effects. The non-drug strategy intravascular laser irradiation of blood (iPBM) has been advocated to improve pain, wound healing, blood circulation, and blood cell function to relieve insomnia and muscle soreness symptoms. Therefore, we assessed whether iPBM improves blood parameters and compared drug use before and after iPBM therapy. Methods: Consecutive patients who received iPBM therapy between January 2013 and August 2021 were reviewed. The associations between laboratory data, pharmacotherapies, and iPBM therapy were retrospectively analyzed. We compared patient characteristics, blood parameters, and drug use within the three months before the first treatment and the three months after the last treatment. We also compared the changes before and after treatment in patients who received ≥10 or 1-9 iPBM treatments. Result: We assessed 183 eligible patients who received iPBM treatment. Of them, 18 patients reported insomnia disturbance, and 128 patients reported pain in any part of their body. After the treatment, HGB and HCT significantly increased after treatment in both the ≥10 and 1-9 iPBM treatment groups (HGB p < 0.001 and p = 0.046; HCT p < 0.001 and p = 0.029, respectively). Pharmacotherapy analysis revealed no significant differences in drug use before and after treatment, though drug use tended to decrease after iPBM. Conclusions: iPBM therapy is an efficient, beneficial, and feasible treatment that increases HGB and HCT. While the results of this study do not support the suggestion that iPBM reduces drug use, further larger studies using symptom scales are needed to confirm the changes in insomnia and muscle soreness after iPBM treatment.
Context Photobiomodulation therapy (PBMT) applied as a preconditioning treatment before exercise has been shown to attenuate fatigue and improve skeletal muscle contractile function during high-intensity resistance exercise. Practical implications for preconditioning muscle with PBMT prior to fatiguing exercise include a safe and non-invasive means to enhance performance and reduce the risk of musculoskeletal injury. Objective To examine the muscle fatigue attenuating effects of PBMT on performance of the shoulder external rotator muscle group when applied as a preconditioning treatment before high-intensity, high-volume resistance exercise. Design Sham-controlled, cross-over design. Setting Laboratory. Participants Twenty healthy men (n=8) and women (n=12) between the age of 18 and 30. Intervention PBMT was administered using a near-infrared laser (λ=810/980nm, 1.8 W/cm2, treatment area = 80cm2-120 cm2) to the shoulder external rotator muscles at a radiant exposure of 10 J/cm2. Subjects performed 12 sets of isokinetic shoulder exercise. Each set consisted of 21 concentric contractions of internal and external rotation at 60°/s. The sets were subdivided into 3 blocks of exercise [Block 1: sets 1-4; Block 2: sets 5-8; Block 3: sets 9-12]. Main Outcome Measures normalized peak torque [Nm/kg], average peak torque [Nm], total work [Nm], and average power [W]. Results During the last block of exercise (sets 9-12), all performance measures for the active PBMT condition were 6.2% to 10% greater than the sham PBMT values (p < 0.02 to 0.001). Conclusions PBMT attenuated fatigue and improved muscular performance of the shoulder external rotators in the latter stages of strenuous resistance exercise.
Background The appearance of the scalp and hair is very important aesthetically regardless of age or sex. Although there are many drugs and treatment methods for scalp problems and hair loss, the treatment response is still insufficient. Aims and Objectives To evaluate the efficacy of low-level light therapy in a helmet-like device. Materials and Methods This study was designed as a 24-week trial with 50 participants. All participants used a helmet-shaped device emitting 630–690, 820–880, and 910–970 nm light wavelengths, for 20 minutes, daily for 24 weeks. A phototrichogram for hair density and thickness, Global Aesthetic Improvement Scale score, erythema index, and sebum secretions of the scalp were evaluated at baseline and at 12 and 24 weeks. Results After 24 weeks of treatment, hair density and hair thickness were found to have significantly increased (P <.01 and P =0.013, respectively) and sebum secretion of vertex area had decreased significantly (P <.01). Of 49 participants, 73.47% of the participants showed improvement in the overall appearance of the scalp (n = 36). Conclusion A helmet-like low-level light therapy device can improve the appearance of the hair, with thickening and increase in the density of the hair, and can improve scalp condition by decreasing sebum secretion.
An ischemic stroke typically accompanies numerous disorders ranging from somatosensory dysfunction to cognitive impairments, inflicting patients with various neurologic symptoms. Among pathologic outcomes, post-stroke olfactory dysfunctions are frequently observed. Despite the well-known prevalence, therapy options for such compromised olfaction are limited, likely due to the complexity of olfactory bulb architecture, which encompasses both the peripheral and central nervous systems. As photobiomodulation (PBM) emerged for treating ischemia-associated symptoms, the effectiveness of PBM on stroke-induced impairment of olfactory function was explored. Novel mouse models with olfactory dysfunctions were prepared using photothrombosis (PT) in the olfactory bulb on day 0. The post-PT PBM was performed daily from day 2 to day 7 by irradiating the olfactory bulb via an 808 nm laser with a fluence of 40 J/cm2 (325 mW/cm2 for 2 min per day). The buried food test (BFT) was used to score behavioral acuity in food-deprived mice to assess the olfactory function before PT, after PT, and after PBM. Histopathological examinations and cytokine assays were performed on the mouse brains harvested on day 8. The results from BFT were specific to an individual, with positive correlations between the baseline latency time measured before PT and its alteration at the ensuing stages for both the PT and PT + PBM groups. Also, the correlation analysis in both groups showed highly similar, significant positive relationships between the early and late latency time change independent of PBM, implicating a common recovery mechanism. Particularly, PBM treatment accelerated the recovery of impaired olfaction following PT by suppressing inflammatory cytokines and enhancing both glial and vascular factors (e.g., GFAP, IBA-1, and CD31). PBM therapy during the acute phase of ischemia improves the compromised olfactory function by modulating microenvironments and inflammation status of the affected tissue.
Photobiomodulation therapy (PBMt) combined or not with oral hypoglycemic medication has not been investigated in type 2 diabetes (T2DM) patients. All ten T2DM patients were assessed randomly at six different occasions (3 with and 3 without regular oral hypoglycemic medication). Capillary glycemia was assessed after overnight fast (pre-prandial), 1h postprandially (standardized meal, 300 kcal), and 30 min, 3h, 6h, 12h post PBMt (830nm; 25 arrays of LEDs, 80mW/array). Three doses (0J - sham, 100J, 240J per site) were applied bilaterally on quadriceps femoris muscles, hamstrings, triceps surae, ventral upper arm and forearm; and randomly combined or not with oral hypoglicemic medication, totaling six different therapies applied for all ten TDM2 patients (PBMt sham, PBMt 100J, PBMt 240J, PBMt sham+medication, PBMt 100J+medication, PBMt 240J+medication). Cardiac autonomic control was assessed by heart rate variability (HRV) indices. Without medication, there was reduction in glycemia after all PBMt doses, with 100J as the best dose that persisted until 12 hours and presented lower area under the curve (AUC). With medication, glycemia decreased similarly among doses. No differences between 100J and sham+medication, but AUC was significantly lower after 100J, suggesting better glycemic control. Low frequency component of HRV increased after sham+medication and 100J, suggesting higher sympathetic activation. PBMt showed time- and dose-response effect to reduce glycemia in T2DM patients. Effects on HRV were consistent with glycemic control. This article is protected by copyright. All rights reserved.
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Modulation of cytochrome c oxidase activity has been pointed as a possible key mechanism for low-level laser therapy (LLLT) in unhealthy biological tissues. But recent studies by our research group with LLLT in healthy muscles before exercise found delayed skeletal muscle fatigue development and improved biochemical status in muscle tissue. Therefore, the aim of this study was to evaluate effects of different LLLT doses and wavelengths in cytochrome c oxidase activity in intact skeletal muscle. In this animal experiment, we irradiated the tibialis anterior muscle of rats with three different LLLT doses (1, 3, and 10 J) and wavelengths (660, 830, and 905 nm) with 50 mW power output. After irradiation, the analyses of cytochrome c oxidase expression by immunohistochemistry were analyzed at 5, 10, 30 min and at 1, 2, 12, and 24 h. Our results show that LLLT increased (p < 0.05) cytochrome c oxidase expression mainly with the following wavelengths and doses: 660 nm with 1 J, 830 nm with 3 J, and 905 nm with 1 J at all time points. We conclude that LLLT can increase cytochrome c oxidase activity in intact skeletal muscle and that it contributes to our understanding of how LLLT can enhance performance and protect skeletal muscles against fatigue development and tissue damage. Our findings also lead us to think that the combined use of different wavelengths at the same time can enhance LLLT effects in skeletal muscle performance and other conditions, and it can represent a therapeutic advantage in clinical settings.
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This study aimed to evaluate the effects of low-level laser therapy (LLLT) immediately before tetanic contractions in skeletal muscle fatigue development and possible tissue damage. Male Wistar rats were divided into two control groups and nine active LLLT groups receiving one of three different laser doses (1, 3, and 10 J) with three different wavelengths (660, 830, and 905 nm) before six tetanic contractions induced by electrical stimulation. Skeletal muscle fatigue development was defined by the percentage (%) of the initial force of each contraction and time until 50 % decay of initial force, while total work was calculated for all six contractions combined. Blood and muscle samples were taken immediately after the sixth contraction. Several LLLT doses showed some positive effects on peak force and time to decay for one or more contractions, but in terms of total work, only 3 J/660 nm and 1 J/905 nm wavelengths prevented significantly (p < 0.05) the development of skeletal muscle fatigue. All doses with wavelengths of 905 nm but only the dose of 1 J with 660 nm wavelength decreased creatine kinase (CK) activity (p < 0.05). Qualitative assessment of morphology revealed lesser tissue damage in most LLLT-treated groups, with doses of 1-3 J/660 nm and 1, 3, and 10 J/905 nm providing the best results. Optimal doses of LLLT significantly delayed the development skeletal muscle performance and protected skeletal muscle tissue against damage. Our findings also demonstrate that optimal doses are partly wavelength specific and, consequently, must be differentiated to obtain optimal effects on development of skeletal muscle fatigue and tissue preservation. Our findings also lead us to think that the combined use of wavelengths at the same time can represent a therapeutic advantage in clinical settings.
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Objective: This study aims to investigate the effects of low-level laser therapy (LLLT) on biceps brachi muscular fatigue in 20 young females. Background data: Exhausting physical activity leads to muscular fatigue, which could decrease muscular strength, and may cause impairment in motor control and muscle pain. Several biochemical and biophysical resources have been studied in an attempt to accelerate the recovery of muscle fatigue. Among these, LLLT is emphasized. Methods: Twenty subjects were randomized in one laser group and one placebo group in two sessions of a crossover design experimental procedure; the second session taking place within 7 days of the first. In the first session, subjects underwent a collection of surface electromyographic (SEMG) data of the biceps brachii muscle, followed by active or placebo LLLT at the same muscle, followed then by another EMG sample of biceps brachii. Blood samples were collected five times during the experimental procedure. Second session procedures were identical to the first, with exception of LLLT, which was the opposite of the first session. The fatigue protocol consisted of 60 sec of elbow flexion-extension movement performed with 75% of one maximum repetition. Blood lactate, EMG fatigue, and the number of elbow flexion-extension repetitions during the fatigue protocol were used to evaluate the effects of laser therapy (808 nm wavelength, 100 mW output power, power density of 35.7 W/cm(2), 70 sec each point and 7 J/point on eight points). Results: No statistical differences were found for eletromyographic fatigue and blood lactate values between groups. Mean numbers of elbow flexion-extension repetitions were 22.6 ± 7.58 after placebo, and 25.1 ± 9.89 after active LLLT group, but these differences were not statistically significant (p=0.342). Conclusions: LLLT had limited effects on delaying muscle fatigue in a young female sample, although a tendency was observed in the active laser group toward showing lower electromyography fatigue of biceps brachii muscle. No intergroup differences were found in the number of muscle contractions and lactate concentration.
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Recent studies have explored if phototherapy with low-level laser therapy (LLLT) or narrow-band light-emitting diode therapy (LEDT) can modulate activity-induced skeletal muscle fatigue or subsequently protect against muscle injury. We performed a systematic review with meta-analysis to investigate the effects of phototherapy applied before, during and after exercises. A literature search was performed in Pubmed/Medline database for randomized controlled trials (RCTs) published from 2000 through 2012. Trial quality was assessed with the ten-item PEDro scale. Main outcome measures were selected as: number of repetitions and time until exhaustion for muscle performance, and creatine kinase (CK) activity to evaluate risk for exercise-induced muscle damage. The literature search resulted in 16 RCTs, and three articles were excluded due to poor quality assessment scores. From 13 RCTs with acceptable methodological quality (≥6 of 10 items), 12 RCTs irradiated phototherapy before exercise, and 10 RCTs reported significant improvement for the main outcome measures related to performance. The time until exhaustion increased significantly compared to placebo by 4.12 s (95 % CI 1.21-7.02, p < 0.005) and the number of repetitions increased by 5.47 (95 % CI 2.35-8.59, p < 0.0006) after phototherapy. Heterogeneity in trial design and results precluded meta-analyses for biochemical markers, but a quantitative analysis showed positive results in 13 out of 16 comparisons. The most significant and consistent results were found with red or infrared wavelengths and phototherapy application before exercises, power outputs between 50 and 200 mW and doses of 5 and 6 J per point (spot). We conclude that phototherapy (with lasers and LEDs) improves muscular performance and accelerate recovery mainly when applied before exercise.
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The use of low level laser (light) therapy (LLLT) has recently expanded to cover areas of medicine that were not previously thought of as the usual applications such as wound healing and inflammatory orthopedic conditions. One of these novel application areas is LLLT for muscle fatigue and muscle injury. Since it is becoming agreed that mitochondria are the principal photoacceptors present inside cells, and it is known that muscle cells are exceptionally rich in mitochondria, this suggests that LLLT should be highly beneficial in muscle injuries. The ability of LLLT to stimulate stem cells and progenitor cells means that muscle satellite cells may respond well to LLLT and help muscle repair. Furthermore the ability of LLLT to reduce inflammation and lessen oxidative stress is also beneficial in cases of muscle fatigue and injury. This review covers the literature relating to LLLT and muscles in both preclinical animal experiments and human clinical studies. Athletes, people with injured muscles, and patients with Duchenne muscular dystrophy may all benefit.
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Background data: Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Methods: Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). Results: LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. Conclusions: LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.
Context Recently, researchers have shown that phototherapy administered to skeletal muscle immediately before resistance exercise can enhance contractile function, prevent exercise-induced cell damage, and improve postexercise recovery of strength and function. Objective To critically evaluate original research addressing the ability of phototherapeutic devices, such as lasers and light-emitting diodes (LEDs), to enhance skeletal muscle contractile function, reduce exercise-induced muscle fatigue, and facilitate postexercise recovery. Data Sources We searched the electronic databases PubMed, SPORTDiscus, Web of Science, Scopus, and Rehabilitation & Physical Medicine without date limitations for the following key words: laser therapy, phototherapy, fatigue, exercise, circulation, microcirculation, and photobiomodulation. Study Selection Eligible studies had to be original research published in English as full papers, involve human participants, and receive a minimum score of 7 out of 10 on the Physiotherapy Evidence Database (PEDro) scale. Data Extraction Data of interest included elapsed time to fatigue, total number of repetitions to fatigue, total work performed, maximal voluntary isometric contraction (strength), electromyographic activity, and postexercise biomarker levels. We recorded the PEDro scores, beam characteristics, and treatment variables and calculated the therapeutic outcomes and effect sizes for the data sets. Data Synthesis In total, 12 randomized controlled trials met the inclusion criteria. However, we excluded data from 2 studies, leaving 32 data sets from 10 studies. Twenty-four of the 32 data sets contained differences between active phototherapy and sham (placebo-control) treatment conditions for the various outcome measures. Exposing skeletal muscle to single-diode and multidiode laser or multidiode LED therapy was shown to positively affect physical performance by delaying the onset of fatigue, reducing the fatigue response, improving postexercise recovery, and protecting cells from exercise-induced damage. Conclusions Phototherapy administered before resistance exercise consistently has been found to provide ergogenic and prophylactic benefits to skeletal muscle.