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Photobiomodulation also known as low-level laser (or light) therapy (LLLT), has been known for almost 50 years but still has not gained widespread acceptance, largely due to uncertainty about the molecular, cellular, and tissular mechanisms of action. However, in recent years, much knowledge has been gained in this area, which will be summarized in this review. One of the most important chromophores is cytochrome c oxidase (unit IV in the mitochondrial respiratory chain), which contains both heme and copper centers and absorbs light into the near-infrared region. The leading hypothesis is that the photons dissociate inhibitory nitric oxide from the enzyme, leading to an increase in electron transport, mitochondrial membrane potential, and adenosine triphosphate production. Another hypothesis concerns light-sensitive ion channels that can be activated allowing calcium (Ca2+) to enter the cell. After the initial photon absorption events, numerous signaling pathways are activated via reactive oxygen species, cyclic AMP, NO, and Ca2+, leading to activation of transcription factors. These transcription factors can lead to increased expression of genes related to protein synthesis, cell migration and proliferation, anti-inflammatory signaling, anti-apoptotic proteins, and antioxidant enzymes. Stem cells and progenitor cells appear to be particularly susceptible to LLLT.
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7000417 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 22, NO. 3, MAY/JUNE 2016
Proposed Mechanisms of Photobiomodulation
or Low-Level Light Therapy
Lucas Freitas de Freitas and Michael R Hamblin
(Invited Paper)
Abstract—Photobiomodulation also known as low-level laser (or
light) therapy (LLLT), has been known for almost 50 years but still
has not gained widespread acceptance, largely due to uncertainty
about the molecular, cellular, and tissular mechanisms of action.
However, in recent years, much knowledge has been gained in this
area, which will be summarized in this review. One of the most
important chromophores is cytochrome c oxidase (unit IV in the
mitochondrial respiratory chain), which contains both heme and
copper centers and absorbs light into the near-infrared region. The
leading hypothesis is that the photons dissociate inhibitory nitric
oxide from the enzyme, leading to an increase in electron trans-
port, mitochondrial membrane potential, and adenosine triphos-
phate production. Another hypothesis concerns light-sensitive ion
channels that can be activated allowing calcium (Ca2+)toenter
the cell. After the initial photon absorption events, numerous sig-
naling pathways are activated via reactive oxygen species, cyclic
AMP, NO, and Ca2+, leading to activation of transcription fac-
tors. These transcription factors can lead to increased expression
of genes related to protein synthesis, cell migration and prolif-
eration, anti-inflammatory signaling, anti-apoptotic proteins, and
antioxidant enzymes. Stem cells and progenitor cells appear to be
particularly susceptible to LLLT.
Index Terms—Low level light therapy, mechanism, mitochon-
dria, cytochrome c oxidase (Cox), Photobiomodulation, Light sen-
sitive ion channels.
I. HISTORICAL INTRODUCTION
THE first evidence of the action of low-level laser irradia-
tion came from the experiments of Dr. Endre Mester, at the
Semmelweis Medical University (Hungary) in 1967. The exper-
iment consisted of shaving the back of mice and implanting a
tumor via an incision in the skin. Mester applied light from a
ruby laser (694 nm) in an attempt to repeat one of the exper-
iments described by McGuff in Boston [1]. McGuff had used
the newly discovered ruby laser to cure malignant tumors both
Manuscript received August 13, 2015; revised March 28, 2016; accepted
April 19, 2016. Date of current version June 10, 2016. The work of M. R.
Hamblin was supported by US NIH under Grant R01AI050875. The work of
Lucas Freitas de Freitas was supported by Fundac¸˜
ao de Amparo `
a Pesquisa do
Estado de S˜
ao Paulo - FAPESP.
L. F. de Freitas is with the Programa de P´
os-Graduac¸˜
ao Interunidades Bio-
engenharia, University of S˜
ao Paulo, S˜
ao Carlos - SP Brazil, and also with
the Wellman Center for Photomedicine, Harvard Medical School, Boston, MA
02114 USA (e-mail: lucasfreitas@usp.br).
M. R. Hamblin is with the Wellman Center for Photomedicine, Harvard Med-
ical School, Boston, MA 02114 USA, the Department of Dermatology, Harvard
Medical School, Boston, MA 02115 USA, and also with the M. R. Hamblin
Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA
02139 USA (e-mail: Hamblin@helix.mgh.harvard.edu).
Color versions of one or more of the figures in this paper are available online
at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JSTQE.2016.2561201
in rats and also tested it in human patients. Unfortunately (or
perhaps fortunately for scientific discovery), Mester’s laser had
only a small fraction of the power possessed by McGuff’s laser.
Therefore Mester failed to cure any tumors, but did observe a
faster rate of hair growth in the treated mice compared to the
controls [2], calling this effect “laser biostimulation”. He later
used a HeNe laser (632.8 nm) to stimulate wound healing in
animals, as well as in clinical studies [3]. For several decades,
the profession believed that coherent laser light was necessary,
but as of today, non-coherent light sources such as light emit-
ting diodes (LED) have proved to be just as efficient as lasers in
promoting photobiomodulation (PBM) [4].
Low-level light therapy (LLLT) or PBM consists of the ap-
plication of light with the purpose of promoting tissue re-
pair, decreasing inflammation, and producing analgesia, usually
using a low-power light source (laser or LED) [5]. Because
of the low power, (usually below 500 mW depending on the tar-
get tissue) the treatment causes no evident temperature rise in the
treated tissue and, therefore, no significant change in the gross
tissue structure [6]. PBM/LLLT differs from other light-based
treatments because it does not ablate and is not based on heat-
ing. It also differs from photodynamic therapy (PDT), which is
based on the effect of light to excite exogenously delivered chro-
mophores to produce toxic reactive oxygen species (ROS) [7].
With the advantage of being non-invasive, the applications of
PBM are broad, going from pain relief to promoting the recovery
of tendinopathies, nerve injuries, osteoarthritis and wound heal-
ing. The complete mechanism of action is still elusive, but the
knowledge that has been gained so far is the subject of the present
review. The importance of parameters in PBM will be discussed,
together with the possible chromophores or photoacceptors, sig-
naling molecules produced after photon absorption, transcrip-
tion factors that may be activated to account for the lasting
effects of a brief light exposure, downstream effector molecules
that follow on, and specific mechanisms that may be applicable
to the different cells and tissues being treated with PBM.
II. PARAMETERS OF PBM
The light parameters and the doses applied are fundamental
in PBM. The most important parameters regarding the light
source and the light doses are described on the following tables
(Tables I and II, respectively):
Low level light therapy refers to the use of light in the red
or near-infrared (NIR) region, with wavelengths usually in the
range of 600 to 700 nm and 780 to 1100 nm, and the laser or
LEDs typically having an irradiance or power density between
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DE FREITAS AND HAMBLIN: PROPOSED MECHANISMS OF PHOTOBIOMODULATION OR LOW-LEVEL LIGHT THERAPY 7000417
TAB L E I
DESCRIPTION OF THE IRRADIATION PARAMETERS
IRRADIATION PARAMETERS
Irradiation Parameter Measurement unit Description
Wavelength nm Light is an electromagnetic form of energy with a wave-like behavior. Its
wavelength is measured in nanometers (nm), and it is visible within the
400–700 nm range.
Irradiance W cm2It can also be called Power Density or Intensity, and corresponds to the
power (in W) divided by the area (in cm2).
Pulse Structure Peak Power (W) Pulse frequency (Hz) Pulse width (s) Duty cycle (%) If the beam is pulsed, the Power should be called Average Power, which
is calculated as follows: Average Power (W) =Peak Power (W) x pulse
width (s) x pulse frequency (Hz)
Coherence Coherence length depends on spectral bandwidth Coherent light produces laser speckle, which is believed to play an
important role on photobiomodulation interaction with cells and
organelles.
Polarization Linear polarized or circular polarized Polarized light is known to lose its polarity in highly scattering media
such as biological tissues, therefore this property is not considered very
often on the effects of PBM.
TAB L E I I
DESCRIPTION OF THE LIGHT DOSE PARAMETERS
LIGHT DOSE PARAMETERS
Irradiation Parameter Measurement unit Description
Energy Joules (J) It cannot be mistook as dose, as it assumes reciprocity (the inverse relationship
between power and time). It is calculated as:
Energy (J) =Power (W) x Time (s)
Energy Density J cm2This is nimportant descriptor of dose, but it could be unreliable when we consider
that it assumes a reciprocity relationship between irradiance and time.
Irradiation Time s Possibly the best way to prescribe and to record PBM would be to define the four
parameters of Table I and then define the irradiation time as the real “dose”.
Treatment Interval Hours, days or weeks Different time intervals may result in different outcomes, but more data need to be
gathered in order to define the extent of the differences between them.
5mWcm
2to 5 W cm2. This type of irradiation can be a
continuous wave or a pulsed light consisting of a relatively low-
density beam (0.04 to 50 J cm2), but the output power can vary
widely from 1 mW up to 500 mW in order not to allow thermal
effects [8]. The wavelength range between 700 and 780 nm has
been found to be rather ineffective as it coincides with a trough
in the absorption spectrum of cytochrome c oxidase (Cox) (see
later). Moreover red/NIR light is chosen because its penetration
through tissue is maximal in this wavelength range, due to lower
scattering and absorption by tissue chromophores. Although for
many years it was thought that the monochromatic nature and
coherence of laser light provided some sort of added benefit
over non-coherent LED light, this view is no longer widely
held. Continuous or pulsed light sources have both been used.
The studies performed for PBM on acute pain and pre-operative
analgesia show that a single treatment (usually only 30–60 sec-
onds) is enough to cause analgesia, while for chronic pain and
some degenerative conditions, more sessions are required [5].
It is known that if the incorrect parameters are applied, the
treatment is likely to be ineffective. There is a biphasic dose re-
sponse curve (or the phenomenon known as hormesis) in which
when too low or too high doses (fluence (J/cm2), irradiance
(mW/cm2), delivery time, or number of repetitions) can lead to
no significant effect or, sometimes, excessive light delivery can
lead to unwanted inhibitory effects [8], [9]. This biphasic re-
sponse follows the “Arndt-Schulz Law” (which states that weak
stimuli slightly accelerate vital activity, stronger stimuli raise it
further until a peak is reached, whereas even stronger stimuli
suppress it until a negative response is achieved), and has been
demonstrated several times in low level light works [10]–[16].
For instance, Bolton irradiated macrophages with the same
energy density (in J cm2) but with different irradiances
(W cm2), and observed different results between the two con-
ditions [17]; Karu and Kolyakov, in 2005, found that the stimu-
lation of DNA synthesis rate is dependent on light intensity at a
constant energy density of 0.1 J cm2with a clear maximum at
0.8 mW cm2[18]; Orion and co-workers worked with a con-
stant energy density and different irradiances on an infarct model
in rats after induced heart attack, and found that the beneficial
effects were obtained at 5 mW cm2, while with irradiances as
low as 2.5 mW cm2or as high as 25 mW cm2there were
significantly less effects [11]; finally, Lanzafame and collabora-
tors used a fixed energy density of 5 J cm2and variable irradi-
ances, ranging from 0.7 to 4 mW cm2, observing that only with
8mWcm
2there were improvements on pressure ulcers in the
treated mice [10].
There were some studies with constant irradiance and vary-
ing fluences. al-Watban and Andres, for instance, observed the
effects of He-Ne laser on the proliferation of Chinese ham-
ster ovary and human fibroblast. The light was delivered at a
7000417 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 22, NO. 3, MAY/JUNE 2016
constant irradiance of 1.25 mW cm2, and a biphasic dose re-
sponse was found with a peak at 0.18 J cm2[19]. Zhang and
collaborators also found a biphasic dose response when they
observed a maximum increase in human fibroblast cells after
irradiation of light at 628 nm with fluence of 0.88 J·cm2, while
there was a marked reduction in the proliferation rate at 9 J cm2
[20].
Regarding the time interval between treatments, Brondon and
colleagues found that the best results for human HEP-2 and
murine L-929 cells proliferation rates were achieved with two
treatments per day, in comparison with one or four treatments per
day. They used an LED with light at 670 nm and irradiance fixed
at 10 mW·cm2, and each treatment consisted on the delivery
of 5 J cm2(the course was stopped after 50 J cm2had been
delivered) [21].
There are also some systematic reviews and meta analyses
of randomized, double-blind, placebo-controlled, clinical trials
(RCTs) available in the literature. We can give as an example
the review from Bjordal, who identified 14 RCTs of suitable
methodological quality. 4 of them failed to report significant
effects because the irradiance was either too low or too high,
or because there was an insufficient delivery of energy [22].
Another review was performed by Tumilty with 25 RCTs of
tendinopathies, 55% of which failed to produce positive out-
comes because of an excessive irradiance delivery in compari-
son with the guidelines set by the World Association for Laser
Therapy [23].
As we have seen, at low doses (up to 2 J cm2), PBM stimu-
lates proliferation, whereas at higher doses (16 J cm2or higher)
PBM is suppressive, pointing to the dose dependence of biolog-
ical responses after light exposure [24]. Other authors, however,
have observed stimulating effects outside the cited range [25],
[26]. A number of different laser light sources, including helium-
neon, ruby, and galliumaluminum-arsenide, have been used to
deliver PBM in different treatments and on different schedules.
Many researchers fail to consider the importance of selecting
the optimum parameters, or they do not have the necessary in-
strumentation or trained personnel to measure them accurately,
resulting in treatment failures. Another cause of failure occurs
whenever the terms are misused or wrongly reported. For in-
stance, energy (J) or energy density (J cm2) are both usually
referred to as “dose”, but they are, in fact, different calculations,
as demonstrated in Table II [27].
III. MOLECULAR MECHANISMS OF PBM
A. Chromophores
1) Cytochrome c Oxidase: Cox is the terminal enzyme of the
electron transport chain, mediating the electron transfer from cy-
tochrome c to molecular oxygen. Several lines of evidence show
that Cox acts as a photoacceptor and transducer of photosignals
in the red and near-infrared (NIR) regions of the light spectrum
[28]. It seems that PBM increases the availability of electrons
for the reduction of molecular oxygen in the catalytic center of
Cox, increasing the mitochondrial membrane potential (MMP)
and the levels of adenosine triphosphate (ATP), cyclic adenosine
monophosphate (cAMP) and ROS as well [29].
PBM increases the activity of complexes I, II, III, IV and
succinate dehydrogenase in the electron transfer chain. Cox is
known as complex IV and, as mentioned before, appears to
be the primary photoacceptor. This assumption is supported
by the increased oxygen consumption during low-level light
irradiation (the majority of the oxygen consumption of a cell
occurs at complex IV in the mitochondria), and by the fact that
sodium azide, a Cox inhibitor, prevents the beneficial effect
of PBM. Besides ATP and cAMP, nitric oxide (NO) level is
increased, either by release from metal complexes in Cox (Cox
has two heme and two copper centers) or by up-regulation of
Cox activity as a nitrite reductase [30].
In fact, it was proposed that PBM might work through the
photodissociation of NO from Cox, thereby reversing the mito-
chondrial inhibition of cellular respiration due to excessive NO
binding [31]. NO is photodissociated from its binding sites on
the heme iron and copper centers from Cox, where it competes
with oxygen and reduces the necessary enzymatic activity. This
allows an immediate influx of oxygen and, thus, the resump-
tion of respiration and generation of ROS. NO can also be
photo-released from other intracellular sites, such as nitrosy-
lated hemoglobin and myoglobin [32].
2) Retrograde Mitochondrial Signaling: One of the most ac-
cepted mechanisms for light-cell interaction was proposed by
Karu [33], referring to the retrograde mitochondrial signaling
that occurs with light activation in the visible and infrared range
(Fig. 1). According to Karu, the first step is the absorption of
a photon with energy hνby the chromophore Cox. This in-
teraction increases MMP (ΔΨm), causing an increase in the
synthesis of ATP and changes in the concentrations of ROS,
calcium (Ca2+) and NO. Furthermore, there is a communica-
tion between mitochondria and the nucleus, driven by changes
in the mitochondria ultrastructure, i.e. changes in the fission-
fusion homeostasis in a dynamic mitochondrial network. The
alteration in the mitochondrial ultrastructure induces changes in
ATP synthesis, in the intracellular redox potential, in the pH and
in cAMP levels. Activator protein-1 and NF-κB have their activ-
ities altered by changes in membrane permeability and ion flux
at the cell membrane. Some complementary routes were also
suggested by Karu, such as the direct up-regulation of some
genes [34].
3) Light Sensitive Ion Channels: The most well-known ion
channels that can be directly gated by light are the channel-
rhodopsins, which are seven-transmembrane-domain proteins
that can be naturally found in algae providing them with light
perception. Once activated by light, these cation channels open
and depolarize the membrane. They are currently being applied
in neuroscientific research in the new discipline of optogenetics
[35].
However, members of another broad group of ion-channels
are now known to be light sensitive [36]. These channels
are called “transient receptor potential” (TRP) channels as
they were first discovered in a Drosophila mutant [36] and
are responsible for vision in insects. There are now at least
50 different known TRP isoforms distributed amongst seven
subfamilies [37], namely the TRPC (‘Canonical’) subfamily,
the TRPV (‘Vanilloid’), the TRPM (‘Melastatin’), the TRPP
DE FREITAS AND HAMBLIN: PROPOSED MECHANISMS OF PHOTOBIOMODULATION OR LOW-LEVEL LIGHT THERAPY 7000417
Fig. 1. Scheme of mitochondrial retrograde signaling pathways as proposed by Karu. The main pathway is represented by continuous arrows, and the
complementary ones are represented by segmented arrows.
Fig. 2. All the seven subfamilies of transient receptor potential channels
(TRP).
(‘Polycystin’), the TRPML (‘Mucolipin’), the TRPA
(‘Ankyrin’) and the TRPN (‘NOMPC’) subfamilies (see Fig. 2).
A wide range of stimuli modulate the activity of different TRP
such as light, heat, cold, sound, noxious chemicals, mechanical
forces, hormones, neurotransmitters, spices, and voltage. TRP
are Ca2+channels modulated by phosphoinositides [38].
The evidence that light mediated activation of TRP is respon-
sible for some of the mechanisms of action of PBM is somewhat
sparse at present, but is slowly mounting.
Mast cells are known to accumulate at the site of skin wounds,
and there is some degree of evidence suggesting that these cells
play a role in the biological effects of laser irradiation on pro-
moting wound healing. Yang and co-workers demonstrated that
after laser irradiation (532 nm), the intracellular [Ca2+]was in-
creased and, as a consequence, there was a release of histamine.
If the TRPV4 inhibitor, ruthenium red, was used, the histamine
release was blocked, indicating the central role of these channels
in promoting histamine-dependent wound healing after laser ir-
radiation [39].
It seems that TRPV1 ion channels are involved in the degran-
ulation of mast cells and laser-induced mast cell activation. It
was demonstrated that capsaicin, temperatures above 42C and
acidic pH could induce the expression of TRPV1 in oocytes, and
these ion channels can be activated by green light (532 nm) in a
power-dependent manner, although blue and red light were not
able to activate them [40]. Infrared light (2780 nm) attenuates
TRVP1 activation by capsaicin in cultured neurons, decreasing
the generation of pain stimuli. TRPV4 is also attenuated by
laser light, but the antinociceptive effect was less intense, there-
fore the antinociception in this model is mainly dependent on
TRPV1 inhibition [41] The stimulation of neurons with pulsed
infrared light (1,875 nm) is able to generate laser-evoked neu-
ronal voltage variations and, in this case, TRPV4 channels were
demonstrated to be the primary effectors of the chain reaction
activated by the laser [42]. However, these effects after exposure
to light above 1,500 nm might occur due to thermal effects, since
water is the main absorber in this region of infrared spectrum.
If it turns out that green light is primarily needed to activate ion
7000417 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 22, NO. 3, MAY/JUNE 2016
channels then clinical applications may be limited due to lack
of penetration into tissue.
4) Direct Cell-Free Light-Mediated Effects on Molecules:
There have been some scattered reports that light can exert
effects on some important molecules in cell free systems (in
addition to the established effect on Cox). The latent form of
transforming growth factor (TGF-β) beta has been reported to be
activated by light exposure [43]. Copper/Zinc Superoxide dis-
mutase (Cu-Zn-SOD) from bovine erythrocytes that had been
inactivated by exposure to pH 5.9 was reactivated by exposure
to He-Ne laser light (632.7 nm) [44]. The same treatment also
reactivated the heme-containing catalase. Amat et al. showed
that irradiation of ATP in solution by 655 nm or 830 nm light
appeared to produce changes in its enzyme reactivity, fluores-
cence and Mg2+binding capacity [45]. However other workers
were unable to repeat this somewhat surprising result [46].
B. Signaling Molecules
1) Adenosine Triphosphate: An increase in intracellular
ATP is one of the most frequent and significant findings af-
ter PBM both in vitro and in vivo [47]. The stimulated synthesis
of ATP is caused by an increased activity of Cox when activated
by light. According to Ferraresi et al. [48], increased Cox ac-
tivity is the mechanism of enhanced muscle performance when
PBM is carried out before various types of exercises, for exam-
ple. The authors found an increased ATP synthesis after LED
(850 ±20 nm and 630 ±10 nm) therapy in different muscles
(one with a predominantly aerobic metabolism, and other with
mixed aerobic and glycolytic metabolism), just like previous
data from Ferraresi et al. [49].
Extracellular ATP participates in a wide array of signaling
pathways, known as purinergic signaling [50]. Originally dis-
covered by Burnstock [51] as a non-adrenergic, non-cholinergic
neurotransmitter, ATP purinergic signaling is mediated by P2Y
G-protein-coupled receptors, and P2X ligand-gated ion chan-
nels [52]. ATP can be hydrolyzed to adenosine that carries out
signals via the P1 G-protein-coupled receptor [53]. Up to the
present date we are not aware of any studies that specifically
show that extracellular (as opposed to intracellular) ATP or
adenosine can be stimulated by PBM.
2) Cyclic AMP: Several workers have shown an increase
in adenosine-3’,5’-cAMP after PBM [54], [55]. Although it
is tempting to suppose that this increase in cAMP is a direct
consequence of the rise in ATP caused by light, firm evidence
for this connection is lacking. It has been reported that cAMP-
elevating agents, i.e. prostaglandin E2, inhibit the synthesis of
TNF and, therefore, down-regulate the inflammatory process.
Lima and co-authors investigated the signaling pathways re-
sponsible for the anti-inflammatory action of PBM (660 nm,
4.5 J cm2) in lung and airways. They found reduced TNF
levels in the treated tissue, probably because of an increase in
cAMP levels. Furthermore, the authors demonstrated that the
inflammation caused by LPS or by TNF in mice lungs was in-
hibited by cAMP-elevating agents. Rolipram, a cAMP-elevating
agent, acts through inhibition of the enzyme phosphodiesterase,
but it does not share this mechanism with low level light [54].
cAMP exerts its cellular effects via activation of three differ-
ent kinds of sensors: cAMP-dependent protein kinase A (PKA)
which phosphorylates and activates cAMP response element-
binding protein (CREB), which then binds to CRE domain on
DNA and in turn activates genes [56];cyclic nucleotide-gated
channels (CNGC) [57] and exchange proteins directly activated
by cAMP (Epac) [58].
3) Reactive Oxygen Species: It was shown that PBM can
produce mitochondrial ROS leading to activation of the tran-
scription factor nuclear factor kappa B (NF-κB), which can act
as a redox-sensor. The fact that the addition of antioxidants in-
hibits the activation of NF-κB by 810 nm light reinforces this
assumption [59].
ROS are one of the classic “Janus face” mediators; benefi-
cial in low concentrations and harmful at high concentrations;
beneficial at brief exposures and harmful at chronic long-term
exposures [60]. ROS are produced at a low level by normal mi-
tochondrial metabolism [61]. The concept of mitohormesis was
introduced to describe the beneficial of low controlled amounts
of oxidative stress in the mitochondria [62]. However when the
MMP is altered either upwards or downwards, the amount of
ROS is increased. In normal cells, absorption of light by Cox
leads to an increase in MMP and a short burst of ROS is pro-
duced. However when the MMP is low because of pre-existing
oxidative stress [63], excitotoxicity [64], or inhibition of elec-
tron transport [63], light absorption leads to an increase in MMP
towards normal levels and the production of ROS is lowered.
There are many different cellular systems that are designed
by evolution to detect excessive levels of ROS and activate
transcription factors to produce extra levels of antioxidant de-
fenses [65]. Hydrogen peroxide and lipid hydroperoxides [66]
are thought to be the ROS most likely to carry out beneficial
redox signaling by reversible oxidation of cysteine thiols in the
sensor protein.
4) Calcium: Changes in the mitochondrial ultrastructure
may lead to alterations in Ca2+concentration. The increment
might be a result of Ca2+influx from the extracellular environ-
ment and gated by the Ca2+channel TRPV. There is evidence
that cytosolic alkalinization can facilitate the opening of TRPV
channels and, since laser irradiation can induce cellular alka-
linization, PBM could induce TRPV opening and a consequent
Ca2+influx. In mast cells, this Ca2+influx can mediate his-
tamine release [67]. However it is also possible that light can
directly activate TRPV channels as discussed above. It should
be noted that PBM usually leads to an increase in intracellular
Ca2+as shown by fluorescent probes [68]. However when in-
tracellular Ca2+levels have been artificially raised (for instance
by causing excitotoxicity with excess glutamate), then PBM can
produce a drop in intracellular calcium and protect the neurons
from dying [64]. The increase in calcium seen after PBM could
also be a result of the release of Ca2+from intracellular stores
[69].
Calcium-sensitive signaling pathways are too numerous to
cover in detail here, but include calcium sensitive enzymes
like protein kinase C (PKC), calcium-calmodulin dependent
kinase II (CamKII) and calcineurin (CaCN) [70], the extra-
cellular calcium–sensing receptor (CaSR) [71], mitochondrial
DE FREITAS AND HAMBLIN: PROPOSED MECHANISMS OF PHOTOBIOMODULATION OR LOW-LEVEL LIGHT THERAPY 7000417
calcium signaling [72], calcium-sensitive adenylyl cyclase [73],
and many others.
5) Nitric Oxide: As mentioned above, NO is often found
to be produced after PBM [74]. NO is a well-known vasodila-
tor acting via stimulation of soluble guanylate cyclase to form
cyclic-GMP (cGMP). cGMP activates protein kinase G, which
causes reuptake of Ca2+and opening of calcium-activated
potassium channels. The fall in concentration of Ca2+prevents
myosin light-chain kinase (MLCK) from phosphorylating the
myosin molecule, leading to relaxation of the smooth muscle
cells in the lining of blood vessels and lymphatic vessels [75].
There are several other mechanisms by which NO could carry
out signaling pathways, including activation of iron-regulatory
factor in macrophages [76], modulation of proteins such as
ribonucleotide reductase [77] and aconitase [78], stimulating
ADP-ribosylation of glyceraldehyde-3-phosphate dehydroge-
nase [79], and protein sulfhydryl group nitrosylation [80].
C. Activation of Transcription Factors
1) Nuclear Factor Kappa B: NF-κB is a transcription fac-
tor that regulates the expression of various genes related to
many cellular functions, i.e. inflammatory and stress-induced
responses and survival. Its activity is regulated by a negative
feedback mediated by an inhibitor called IκB, which binds to
NF-κB to inactivate it, or can undergo ubiquitination and go to
proteasomal degradation in order to release NF-κB. The tran-
scription factor, then, can be translocated to the nucleus and
promote gene transcription. Several lines of evidence reveal
that NF-κB is redox-sensitive, since ROS can directly activate
it, or alternatively ROS could be involved in indirect activa-
tion of NF-κB via TNF, interleukin-1 (IL-1) and phorbol esters.
PBM can boost ROS generation, and it was shown that light
irradiation can induce NF-κB activation [59].
The increased NF-κB production after PBM stimuli leads to
enhanced gene transcription that leads to reduced cell death, to
cell proliferation, to cell migration [81] and enhanced neurolog-
ical function. Fig. 3 shows an overview of the different groups
of genes that have NF-kB response elements.
If the total energy density delivered is too high, however,
the injury paradoxically tends to be exacerbated by increased
oxidative stress, and an over-abundant activation of NF-κB.
The biphasic dose effects of PBM are thought to occur due to an
excessive generation of ROS, excessive production of NO, to the
activation of some cytotoxic pathways, and to excessive NF-κB
activation [82]. In addition, if the tissue is stressed or ischemic,
mitochondria can synthesize NO that can displace oxygen from
binding to Cox, but this leads to a reduced ATP synthesis and
to an increased oxidative stress that can lead to inflammation
when NF-κB is activated [83].
Classical mitochondrial inhibitors such as rotenone are known
to decrease mitochondrial ATP levels, produce ROS and activate
NF-κB. Low-level light still produces ROS and activates NF-
κB, but in this case increases ATP levels. Antioxidants do not
inhibit this ATP increase, suggesting that light augments the
electron transport and potentially causes electron leakage (in
the absence of antioxidants) and superoxide production [59].
2) Receptor Activator of Nuclear Factor Kappa-B Ligand
(RANKL): RANKL is a transmembrane protein member of the
TNF superfamily, involved in bone regeneration and remodeling
(acting on osteoclast differentiation and activation). It is also a
ligand for osteoprotegerin (OPG). The RANKL/OPG ratio de-
termines whether bone is removed or formed during the remod-
eling process. The remodeling cycle consists in the increase in
the expression of RANKL by osteoblasts, and subsequent bind-
ing to RANK receptor, which is highly expressed on osteoclastic
membrane. This causes an expansion of the osteoclast progen-
itor pool, differentiation into mononucleated progenitor cells,
increased survival, fusion into multinucleated osteoclasts and,
finally, their activation. Osteoblasts can modulate this process
by expressing OPG, which is a secretory soluble receptor and
inhibitor of RANK receptor.
Parenti et al. investigated the RANKL/OPG ratio in
osteoblast-like cells that were irradiated with GaAlAs laser (915
nm) using doses ranging from 1 to 50 J cm2. Although the dif-
ferences were not statistically significant, there was a trend for
a rapid and transitory increase in the RANKL/OPG ratio for all
the tested doses. It seems that this ratio after PBM depends on
the tissue and on the parameters used, since there is evidence
of an increase in RANKL/OPG ratio in human alveolar bone-
derived cells irradiated with 780 nm light, while in rat calvarial
cells irradiated with 650 nm light the results were the opposite
[84].
3) Hypoxia Inducible Factor (HIF-1α): HIF-1αis a protein
involved in cellular adaptation to hypoxia. It is stabilized at low
oxygen tensions, but in the presence of higher oxygen concen-
trations it is rapidly degraded by prolyl hydroxylase enzymes,
which are oxygen-dependent. HIF-1αactivates genes that are
important to the cellular response to hypoxic conditions, such
as vascular endothelial growth factor (VEGF), VEGF-receptor,
glucose carrier (GLUT-1) and phosphoglycerate kinase (PGK)
genes. Since there is no significant changes in gross tissue oxy-
gen concentration during PBM, HIF-1αactivation may be me-
diated by the mitogen-activated protein kinase (MAPK) and
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway,
by growth factors or cytokines [85]. Another possible expla-
nation is that the sudden boost in cellular respiration caused
by light activation of Cox depletes the low amount of oxygen
that is present in hypoxic tissues but which is not being rapidly
consumed because of inhibited electron transport. This sudden
oxygen depletion then rapidly activates HIF-1α.
Cury demonstrated the pro-angiogenic effect of PBM using
660 nm and 780 nm light on skin flaps in rats. He observed that
angiogenesis was induced by an increase in HIF-1αand VEGF
expression, as well as by a decrease in matrix metalloproteinase
2 (MMP-2) activity [85]. Cury observed that only 660 nm light
was able to increase HIF-1αexpression, and although VEGF
induction occurred in all light doses used, only 40 J cm2was
able to induce angiogenesis, as well as an increase MMP-2
activity.
4) Akt/GSK3β/β-Catenin Pathway: Low-level light may
exert a prosurvival effect on cells via the activation of
AKT/GSK3β/β-catenin pathway. Basically, protein kinase B
(also known as AKT) can be activated by LLL irradiation, and
7000417 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 22, NO. 3, MAY/JUNE 2016
Fig. 3. Overview of the different groups of genes and molecules that have NF-kB response elements. In principle these could be activated by NK-kB signaling
pathway triggered by the ROS produced during LLLT.
then interact with glycogen synthase kinase 3β(GSK3β), in-
hibiting its activity. GSK3B is a serine-threoninekinase which
mediates various cellular signaling pathways, exerts metabolic
control, influences embryogenesis, and is involved in cell death
and in oncogenesis. There is evidence that this kinase is involved
in the pathogenesis of Alzheimer’s disease, since it promotes hy-
perphosphorylation of tau protein and causes the formation of
neurofibrillary tangles (NTFs), both classic hallmarks of this
disease.
The decreased activity of GSK3βis due to the fact that PBM-
activated AKT increases the phosphorylation level of its Ser9
residue, which allows the N-terminus of GSK3βto bind with its
own binding site. This leads to an accumulation of β-catenin and
its translocation into the nucleus, where it can exert its prosur-
vival action. β-catenin is an important component of Wnt sig-
nalling pathway, responsible for the inhibition of axin-mediated
β-catenin phosphorylation by GSK3β. This helps to stabilize the
under-phosphorylated form of β-catenin, and ensure that it is no
longer marked for proteasome degradation, so it can accumulate
and travel to the nucleus. Once there, the prosurvival action of
β-catenin relies on the increased TCF/LEF-dependent transcrip-
tional activity. This prosurvival effect can be useful in the treat-
ment of neurodegenerative diseases, such as Alzheimer’s [86].
One of the most important regulators of apoptosis is Bax,
a member of Bcl-2 family. It is translocated from the cytosol
to the mitochondria when a pro-apoptotic stimulus is present,
and this translocation is inhibited by PBM, according to Zhang
et al. The authors hypothesized that GSK3βis the mediator
between Akt and Bax during the PBM anti-apoptotic process.
The authors found that GSK3βinteracts with Bax and activates
it, promoting its translocation directly, but PBM activates Akt
which inhibits the activation of GSK3β, thus inhibiting Bax
translocation. Using inhibitor compounds such as wortmannin
and lithium chloride, there was a significant inhibition of the
anti-apoptotic effect observed after PBM, suggesting that
PI3K/Akt pathway (inhibited by wortmannin) and GSK3β
translocation (inhibited by lithium chloride) play a key role in
the protection against apoptosis caused by low level light. LiCl,
however, was not able to reduce Bax translocation and apoptosis
like PBM, so there must be other upstream regulators of Bax
translocation during apoptosis. In conclusion, PBM exerted a
pro-survival action through selectively activating the PI3K/Akt
pathway and suppressing GSK3β/Bax pathway [87].
5) Akt/mTOR/CyclinD1 Pathway: PBM has been demon-
strated to be useful for stimulating proliferation of normal cells,
but for dysplastic and malignant cells it could be dangerous.
Sperandio et al. provided an example of this situation, observ-
ing that oral dysplastic cells, considered pre-malignant, had
their viability increased after PBM (660 or 780 nm, 2 to 6 J
cm2). Moreover, these workers showed higher expression of
proteins related to cancer progression and invasion, i.e. Akt,
HSP90, pS6ser240/244, and Cyclin D1. The data suggest that
Akt/mTOR/Cyclin D1 pathway was important for this pheno-
type differentiation, since the tested oral cancer cells showed
higher levels of the signaling mediators that are part of this
pathway [88].
6) Extracellular Signal-Regulated Kinase (ERK)/ Forkhead
box protein M1 (FOXM1): FOXM1 is a protein involved in the
regulation of the transition from G1 to S phase of the cell cy-
cle and progression to mitotic division. Ling et al. investigated
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Fig. 4. A model of the signaling pathways for LLLT protecting cell from UVB-induced senescence.
the protective effect of PBM using red light at 632.8 nm against
senescence caused by UV light, and reported an activation of the
ERK/FOXM1 pathway that caused a reduction in the expression
of p21 protein and G1 phase arrest. Senescence was attenuated
by over-expression of FOXM1c with or without PBM, and if
FOXM1 was inhibited by shRNA, the effect of PBM in reduc-
ing cell senescence was abrogated. PBM promoted the nuclear
translocation of ERK, increasing FOXM1 accumulation in the
nucleus and the transactivation of c-Myc and p21 expression.
Inhibition of the mitogen-activated kinase (MEK)/ERK path-
way with an MEK inhibitor PD98059 prevented the nu-
clear translocation of FOXM1 after PBM, suggesting that
Raf/MEK/MAPK/ERK signaling is crucial for the anti-cell
senescence effect of PBM mediated by FOXM1 [89]. Fig. 4
summarizes these findings.
7) Peroxisome Proliferator-Activated Receptors (PPAR)-y:
PPAR are mostly present in airway epithelial cells, but also in
smooth muscle cells, myofibroblasts, endothelial cells of the
pulmonary vasculature and in inflammatory cells such as alve-
olar macrophages, neutrophils, eosinophils, lymphocytes and
mast cells. They are nuclear receptors with transcription fac-
tors that regulate gene expression. PPAR-y is involved in the
generation of heat shock protein 70 (HSP-70), which is anti-
inflammatory, while PPAR-c expression occurs due to an in-
flammatory response and are associated with massive lung in-
jury and neutrophil infiltration in lungs of mice subjected to
endotoxic shock [90].
Lima and co-authors reported a study in which rats were
irradiated with 660-nm light (5.4 J) on the skin over the bronchus
(chest). They observed a marked rise in the expression of PPAR
mRNA after PBM, as well as increased PPAR-y activity in
bronchoalveolar lavage (BALF) cells from animals subjected
to laser treatment. In conclusion, Lima proposed that PBM can
work as a homeostatic facilitator, increasing the expression of a
transcription factor that is signaling the synthesis of HSP70 and
other anti-inflammatory proteins [90].
8) Runt-Related Transcription Factor 2 (RUNX-2): RUNX-
2 is related to osteoblastic differentiation and skeletal morpho-
genesis, acting as a scaffold for nucleic acids and regulatory
factors that are involved in the expression of skeletal-related
genes. It regulates the expression of genes related to extracel-
lular matrix components during bone cell proliferation. PBM
can increase the expression of RUNX-2, contributing to a better
tissue organization, even in diabetic animals as seen by Pa-
troc´
ınio-Silva [91].
D. Effector Molecules
1) Transforming Growth Factor: TGF-βis a strong stimu-
lator of collagen production, inducing the expression of extra-
cellular matrix components and inhibiting its degradation by
inhibiting matrix metalloproteinases (MMPs). TGF-βexpres-
sion is elevated during the initial phase of inflammation after
an injury, and stimulates cellular migration, proliferation and
interactions within the repair zone [92].
Dang and co-workers suggested that TGF-β/SMAD signaling
pathway might play a role in PBM used for non ablative reju-
venation [93]. They found that 800 nm diode laser irradiation
was able to induce collagen synthesis through the activation
of TGF-β/SMAD pathway in a light dose-dependent manner.
40 J cm2was the most effective light dose in enhancing the
gene expression of procollagen type I and IV, compared to 20
and 60 J cm2. The dermal thickness followed the results for
the synthesis of collagen, demonstrating that this process was
indeed dose-dependent [93].
Aliodoust et al. treated rats with 632.8 nm light and observed
increased expression of TGF-β1 (one of the three isoforms of
TGF-β) mRNA. TGF-β1 is responsible for the initial scar tissue
formed at the wound site. It enhances tendon repair during the
fibrosis period via the stimulation of cell proliferation and mi-
gration, as well as the synthesis of collagen and proteoglycans
[92].
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Fig. 5. Reactive oxygen species sensors and signaling.
2) Oxidative Stress: The inflammatory process involves an
increase in ROS and RNS production, accompanied by a re-
duction in the activity of antioxidant defenses, as well as by by
alterations on the activity of inflammatory mediators and signal-
ing molecules, such as described in Fig. 5. This oxidative stress
situation can activate NF-κB, as mentioned before, leading to
modifications in the expression of genes for pro-inflammatory
cytokines, growth factors, chemokines and adhesion molecules.
Assis et al. investigated the effects of PBM on muscle injury
using 808 nm light (1.4 J), and observed reduced lipid peroxi-
dation accompanied by a decreased COX-2 mRNA expression
and an increased SOD mRNA expression after irradiation. There
was a reduced formation of nitrotyrosine, indicating that iNOS
activity was lower and, consequently, NO and peroxynitrite pro-
duction was decreased. In conclusion, the inhibition of oxidative
and nitrosative stress contributed to a decrease in the deleterious
effects observed after muscle injury [94].
3) Pro- and Anti-Inflammatory Cytokines: Many cytokines
and inflammatory mediators have their levels altered by
low-level light irradiation, regardless if they have pro- or anti-
inflammatory actions, i.e. TNF, various interleukins, histamine,
TGF-β, prostaglandins and eicosanoids. It seems that when
inflammation is present, PBM exerts an anti-inflammatory
action, but in the absence of inflammation, PBM provide pro-
inflammatory mediators that could help in tissue remodeling
and to mediate cell function. Wu and co-workers investigated
the photoacceptor role of Cox and found that the excitation of
Cox initiates a photoreaction that results in histamine release in
vitro. The induced signals from mitochondria to cytosol cause
alkalinization of the cytosol, which leads to the opening of
TRPV channels. This results in an increment of [Ca2+]and,
consequently, in an enhanced histamine release [67]. Chen
demonstrated in 2014 that an increased calcium influx occurred
in mast cells after laser irradiation, and this caused histamine
release that could help promoting wound healing. Furthermore,
he found that during short-term muscle remodeling after
cryoinjury, cytokines expression is also modulated by PBM,
leading to a decreased expression of TNF and TGF-β[95].
Although NF-κB activation is known to be pro-inflammatory,
PBM has a pronounced anti-inflammatory activity even with
NF-κB activation. In fact, the anti-inflammatory effects of PBM
could be abrogated if a NF-κB inhibitor is used. This probably
occurs because the initial response to cell stress typical of NF-κB
activation triggers another response to lower NF-κB activation
after PBM had its therapeutic effect. Another possibility is that
the initial pro-inflammatory response induced by PBM leads to
the expression of eicosanoids that are able to decrease and to
end inflammation [95].
4) Brain-Derived Neurotrophic Factor (BDNF): BDNF is
part of the family of neurotrophins, molecules that exert ac-
tions on nerve cells. BDNF, specifically, seems to modulate
dendritic structure and to potentiate synaptic transmission in
the central nervous system. In order to investigate the effects
of low-level light on BDNF levels, Meng et al. treated nerve
cells with 632.8 nm light (doses from 0.5 to 4 J cm2). There
was a regulatory role of PBM in neuroprotection and dendritic
morphogenesis. PBM attenuated the decrease of BDNF, appar-
ently by the ERK/CREB pathway, and this could be useful in
the treatment of neurodegenerative disorders [96].
5) Vascular Endothelial Growth Factor: Angiogenesis is a
complex mechanism, requiring several cell types, mediators and
signaling pathways. It is initiated by cell migration and invasion
of endothelial cells, subsequent lumen formation and connec-
tion of the new vascular segments with pre-existing ones, and
finally, remodeling of extracellular matrix. This remodeling is
dependent on an adequate MMPs activity. VEGF and HIF-1α
are critical to the angiogenic process.
PBM has been reported to induce angiogenesis in several ex-
perimental models. For example, Cury et al. observed a marked
increase in the number of vessels in the skin flap of animals
treated with 660 and 780 nm PBM, alongside with a marked
increase in VEGF mRNA expression [85].
6) Hepatocyte Growth Factor (HGF): HGF is a cytokine
that regulates cell proliferation, motility, morphogenesis and
exerts anti-apoptotic and anti-inflammatory activity during hep-
atic regeneration. The activation of its transmembrane tyrosine
kinase receptor, called Met receptor, leads to autophosphory-
lation of tyrosine residues and phosphorylation of downstream
signaling molecules, such as PI3K and MAPK pathway proteins.
Ara´
ujo and co-workers observed that, after 632.8 nm PBM,
hepatectomized animals showed an increase in the expression
of HGF followed by increased phosphorylation of Met and its
downstream signaling molecules Akt and ERK. This indicates
that PBM could enhance liver regeneration after hepatectomy
[97].
7) Basic Fibroblast Growth Factor (bFGF) and Keratinocyte
Growth Factor (KGF): Growth factors play a key role in the
wound healing process, mediating the transfer of signals be-
tween the epithelium and the connective tissue, especially bFGF
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and KGF. bFGF is known to be a potent mitogen and chemoat-
tractant for endothelial cells and fibroblasts, as well as accel-
erating the formation of granulation tissue and to induce re-
epithelization. KGF is produced by fibroblasts and exerts a
paracrine action on keratinocytes, therefore, it is responsible
for the proliferation and migration of epithelial cells, as well as
for the maintenance of the epithelium normal structure.
When gingival fibroblasts from a primary culture were irra-
diated twice with 660 or 780 nm low-level light in a study from
Damante et al., production of KGF and bFGF was increased.
Red light was more effective in stimulating KGF production,
but no significant change in bFGF production was seen with red
light. NIR light, however, was capable of inducing bFGF release
[98]. These results could explain how PBM can help the wound
healing process.
8) Heat Shock Proteins (HSP): Heat shock protein 27
(HSP27) is an important member of the small HSP family, with
an ATP-independent chaperone activity that is produced in re-
sponse to oxidative stress in order to modulate inflammation and
to regulate the dynamics of the actin cytoskeleton. When HSP27
is activated, it facilitates the phosphorylation of IκB, causing it
to be degraded in the proteasome and increasing NF-κB activity.
It also contributes to the regulation of NO and ROS production,
iNOS expression and TNF secretion. However HSP27 plays
a negative role in TNF-mediated IκB kinase (IKK) activation.
The results of a study performed by Lim and co-workers with
HSP27-silenced cells showed that 635 nm light irradiation was
not able to decrease ROS generation if HSP27 was not present,
indicating that this chaperone plays an important role in ROS
decreasing during inflammation and PBM [99].
HSP70 is part of the normal wound healing process, alongside
IL-6 and TGF-β1. Visible (532 nm) and NIR (815 nm) light have
been demonstrated to induce HSP70 expression in treated skin
cells, and this is important for skin rejuvenation interventions,
since there is a consequent effect consisting on the assistance of
the correct folding and transport of newly synthesized collagen
[93].
HSP90 is another chaperone, which assists the maturation of
Akt enabling it to perform its downstream actions. Increased
activity of chaperones is certainly not desired in cancer, but it
could be useful in healing processes. Sperandio et al. found
higher levels of HSP90 in laser-treated cells, and an isoform of
this chaperone, HSP90N, which has an oncogenic potential, was
found in the experimental groups. This isoform is commonly
overexpressed in tumor tissues and is secreted by advantage
stages of melanoma [88].
E. Cellular Mechanisms
1) Inflammation: Lim and co-workers found that 635 nm
light irradiation at low power can lead to an anti-inflammatory
effect by inhibiting prostaglandin E2 (PGE2) production and
cyclo-oxygenase 1 and 2 (COX-1 and COX-2) mRNA expres-
sion. The light irradiation was able to decrease intracellular
ROS, which mediate the expression of calcium-dependent phos-
pholipase A2 (cPLA2), secretory phospholipase A2 (sPLA2),
and COX-2, and also inhibit the release of PGE2 [99].
PGE2 synthesis is dependent on NF-κB modulation of the
cellular signaling mechanism. NF-κB is found in the cytosol
in its dimeric form of NF-κB/IκB (the latter is an inhibitory
protein). Pro-inflammatory stimuli, such as LPS, are able to
activate the NF-κB upstream signaling regulator IκB kinase
(IKK), responsible for the phosphorylation and degradation of
IκB. The free NF-κB is translocated to the nucleus and induces
the expression of pro-inflammatory genes [8]. Lim demonstrated
that 635 nm light irradiation suppressed the release of PGE2,
possibly through a mechanism related to the inhibition of NF-κB
pathway. It did not affect the phosphorylation of IκB, IKK and
NF-κB in HSP27-silenced human gingival fibroblasts (hGFs),
suggesting that NF-κB modulation by 635 nm light through
HSP27 is required for the down-regulation of pro-inflammatory
gene expression in these fibroblasts [99].
Macrophages are important antigen-presenting cells, and are
involved in the induction of primary immunologic response.
Interferon gamma (IFN-γ) polarization (either via classical or
M1 activation) programs monocytes for increased phagocytic
activity, as well as for anti-tumor activity and allergy suppres-
sion. Recently, Chen reported that 660 nm PBM could promote
M1 polarization of monocytes, and influence the expression of
cytokines and chemokines at the level of mRNA and protein ex-
pression. The effect was dose-dependent, since the optimal light
dose found was that of 1 J cm2, compared to 2 and 3 J cm2.
Furthermore, the author could also clarify the mechanisms of
epigenetic regulation by PBM in immune cells. Modifications
on histones, usually carried out by histone acetyl- or methyl-
transferases, could be induced by PBM: histone H3 and H4
acetylation and H3K4 trimethylation in the TNF gene promoter
area, and histone H3 acetylation in the IP-10 gene promoter
region. M1-related immunoregulation is important for antivi-
ral immunity, antitumor immunity, and for the pathogenesis of
infammation in autoimmune conditions, therefore PBM could
help promoting anti-viral and anti-tumor immunity, but could
enhance autoimmune and rheumatoid diseases [95].
2) Cytoprotection: Studies have shown that PBM in vitro
protects cells at risk from dying due to treatment with vari-
ous different toxins. Methanol, for instance, generates a toxic
metabolite (formic acid) that inhibits Cox. Since PBM enhances
mitochondrial activity via stimulation of Cox, it also promotes
cell survival during formic acid toxicity. This was demonstrated
by Eells, who used red light (670 nm) in a rodent model of
methanol toxicity and found that the light irradiation induced a
significant recovery of cone- and rod-mediated function in the
retina of rats after methanol intoxication, as well as a protection
against histological damage resulting from formic acid [100].
Cyanide is another toxic compound that can have its effects
attenuated by PBM. Potassium cyanide-induced apoptosis of
neurons was decreased with a pretreatment with 670 nm light.
This is explained by the fact that PBM decreased the expression
of caspase-3 (commonly increased by cyanide) and reversed the
cyanide-induced increased expression of Bax, while decreasing
the expression of Bcl-2 and inhibiting ROS generation [101].
Wong-Riley and co-workers show that LED pretreatment was
not able to restore enzymatic activity in cells to control levels
after cyanide toxicity, but it successfully reversed the toxic effect
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of tetrodotoxin, especially with 670 and 830 nm light. These
wavelengths correspond to the peaks in the absorption spectrum
of Cox, suggesting that this photobiomodulation is dependent
of the up-regulation of Cox [102].
PBM can be useful in the treatment of Alzheimer’s disease,
since low-power laser irradiation promotes Yes-associated pro-
tein (YAP) cytoplasmic translocation and amyloid-β-peptide
(Aβ) inhibition. Aβdeposition is a known hallmark of
Alzheimer’s disease, while YAP translocation is involved in the
regulation of Aβ-induced apoptosis. Zhang published a study
demonstrating that 832.8 nm light irradiation is able to reduce
Aβ-induced toxicity by inhibiting apoptosis through the activa-
tion of Akt/YAP/p73 signaling pathway [103].
3) Proliferation: Several cell types can have their prolif-
eration levels increased by PBM. Keratinocytes, for example,
showed an enhanced proliferation after 660 nm light irradia-
tion, accompanied by an increased expression of Cyclin D1 and
a faster maturation of keratinocytes in migration to the wound
sites, via the expression of proteins involved in the epithelial pro-
liferation process, namely p63, CK10 and CK14. This is useful
for the improvement of epithelial healing [104]. Furthermore,
fibroblasts irradiated with 632.8 nm light had their proliferation
stimulated and their cell viability increased, demonstrating the
stimulatory effect of PBM and the usefulness of this therapy in
the wound healing process [105].
Vascular endothelial cells exposed to 635 nm irradiation pro-
liferate faster than non-irradiated cells, showing a decreased
VEGF concentration. This suggests that laser-induced cell pro-
liferation is related to a decrease in VEGF concentration. 830
nm irradiation decreased TGF-βsecretion by the endothelial
cells [106].
Amid et al. published a review about the influence of PBM
on the proliferation of osteoblasts. According to the studies
reviewed by the authors, wavelengths between 600 nm and
1000 nm have been used, and resulted in positive effects on
dentistry, on anti-inflammatory process and on osteoblastic pro-
liferation [107].
Fibroblasts irradiated with 632.8 nm light had their prolifera-
tion stimulated and their cell viability increased, demonstrating
the stimulatory effect of PBM and the usefulness of this therapy
in the wound healing process.
4) Migration: Tendon healing requires migration of teno-
cytes to the injured area, with consequent proliferation and syn-
thesis of extracellular matrix. Tsai and co-workers evaluated the
effect of 660 nm light on rat Achilles tendon-derived tenocytes,
and found that dynamin-2 expression was enhanced and the
migration was stimulated in vitro. Inhibiting dynamin-2 with
dynasore suppressed this stimulatory effect of PBM, leading to
the conclusion that tenocyte migration stimulated by low-level
light was mediated by the up-regulation of dynamin-2 [108].
Other cell types are also influenced by light irradiation.
Melanocytes, for instance, showed an enhanced viability and
proliferation after blue and red light irradiation. Melanocytes
migration was enhanced by UV, blue and red light in lower
doses, but a non-stimulatory effect was observed for higher
light doses. Blue light seemed to be more effective compared to
UV and red lasers [109]. Human epidermal stem cell migration
and proliferation were increased alongside an increased phos-
phorylation of autocrine ERK, which contributed to accelerated
wound re-epithelialization [110]. Finally, 780 nm irradiation
seemed to be able to accelerate fiber sprouting and neuronal
cell migration, at least in embryonic rat brain cultures. Large-
size neurons with a dense branched interconnected network of
neuronal fibers were also observed after laser irradiation. These
results can be seen in Rochkind’s work, and may contribute
for future treatment modalities for neuronal injuries or diseases
[111].
5) Protein Synthesis: As mentioned before, PBM was able
to increase the expression of proteins related to the proliferation
and maturation of epithelial cells: p63, CK10 and CK14 [104]. In
fact, low level light can increase the expression of several other
proteins. A good example is the enhanced collagen I expression
by fibroblasts 2 days after 810 nm light irradiation, as demon-
strated by Frozanfar and co-workers in 2013 [112]. Moreover,
osteoblasts irradiated with 830 nm light increased the expression
of proteins and proteoglycans such as osteoglycin and mimecan.
Osteoglycin is a leucine-rich proteoglycan, once called osteoin-
ductive factor, easily found in bone matrix, cartilage cells and
connective tissues. They play a regulatory role in cell prolifera-
tion, differentiation and adhesion of osteoblastic cells, therefore
PBM applied on the early proliferation stage of osteoblasts are
important for the stimulation of bone formation, in concert with
some growth factors and matrix proteins [113].
6) Stem Cells: It appears that stem cells are particularly sen-
sitive to light. PBM induces stem cell activity shown by in-
creased cell migration, differentiation, proliferation and viabil-
ity, as well as by activating protein expression [114]. Mesenchy-
mal stem cells, usually derived from bone marrow, dental pulp,
periodontal ligament and from adipose tissue, proliferate more
after light irradiation (usually with wavelengths ranging from
600 to 700 nm). Since stem cells in their undifferentiated form
show a lower rate of proliferation, this may be a limiting factor
for the clinical effectiveness of stem cell therapies, PBM of-
fers a viable alternative to promote the translation of stem cell
research into the clinical arena [115].
Min and co-workers reported that the cell viability of adipose-
derived stem cells was found to be increased after irradiation
with 830 nm light. Their in vivo results also revealed elevated
numbers of stem cells compared to the control group [116].
Epidermal stem cells can also be influenced by light, as demon-
strated by Liao et al. The authors reveal that 632.8 nm light has
photobiological effects on cultured human epidermal stem cells,
such as an increase in proliferation and migration in vitro [110].
Soares observed a similar effect on human periodontal ligament
stem cells irradiated with a 660 nm diode laser [117].
F. Tissue Mechanisms
1) Muscles: We already mentioned the positive results for
PBM in muscle recovery, reported by Ferraresi et al. The authors
demonstrated the usefulness of PBM in muscle recovery after
injury. The authors concluded that it takes between 3 and 6 hours
for the PBM to exert maximum effect on the muscle physiology,
consisting of increased matrix metalloproteinase activity and
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ATP synthesis. This effect could still be observed 24 hours after
the laser irradiation [49].
Rochkind and co-workers have also worked with PBM ap-
plied to muscles, investigating the influence of low power laser
irradiation on creatine kinase (CK) and the amount of acetyl-
choline receptors (AChRs) present in intact gastrocnemius mus-
cle in vivo, as well as the synthesis of DNA and of CK in muscle
cells in vitro. The authors found that PBM significantly in-
creased CK activity and AChR level in one and two months,
when compared to control animals. The biochemical changes
on muscle cells might be due to a trophic signal for increased
activity of CK, which leads to a preservation of a reservoir of
high-energy phosphate that is available for rapid ATP synthesis
[118].
2) Brain: Regarding the neurological field, PBM can lead
to cognitive benefits and memory enhancement in case of brain
damage caused by controlled cortical impact (CCI). Khuman
and co-workers found that a 500 mW cm2laser irradiation (60 J
cm2) for two minutes improved spatial learning and memory of
mice with CCI, and this was not observed in sham-injured mice.
The authors observed a brief increase in the temperature of brain,
but it returned to baseline before 5 minutes of irradiation. They
also observed reduction of microgliosis at 48 h. Low level light
can be useful in traumatic brain injury (TBI) treatment, since
suboptimal light doses demonstrated to affect spatial memory,
as assessed by visible platform trials, even in the absence of non-
spatial procedural learning, which is hippocampus-independent
[82].
NIR light exerts a protective effect on neurons, but the mech-
anisms are not fully understood. However, two mechanisms
may be involved, and the first that will be discussed is the di-
rect action of NIR light on the cells, improving mitochondrial
function, reducing inflammation, and helping the brain to re-
pair itself. Xuan et al reported that transcranial NIR light could
stimulate the process of neurogenesis in the hippocampus and
subventricular zone (SVZ) in mice with CCI TBI [119]. These
newly formed neuroprogenitor cells could travel to the injured
region of the cortex to help in the repair of the damaged region.
In another study the same group showed that BDNF was in-
creased in the hippocampus and SVZ at one week post TBI, and
that at 4 weeks post TBI there as an increase in synaptogenesis
in the cortex showing that new connections between existing
brain cells could be stimulated by light [120].
The second mechanism is based on the hypothesis that NIR
can trigger a systemic response, this time not so directly, sug-
gesting the involvement of one or more circulating molecules or
cell types. This assumption is based on studies reporting remote
effects on tissues after irradiation of NIR light on specific sites,
such as skin wounds. Another study reported brain protection
in mice after remote irradiation with NIR light to the dorsum
of the animals, without any direct irradiation on the head. One
possibility to explain these remote effects is the stimulation of
mast cells and macrophages, which could help to protect cells
in the brain, as well as the modulation of inflammatory medi-
ators, like the down-regulation of pro-inflammatory cytokines
and up-regulation of anti-inflammatory cytokines. Another pos-
sibility is the involvement of bone-marrow derived stem cells,
since NIR light can increase the proliferation of c-kit-positive
cells located in the bone marrow of the skull, which are then
recruited to damaged tissues, especially the myocardial infarct
site. These progenitor cells can, alongside with immune cells,
secrete trophic and pro-survival factors such as nerve growth
factor and VEGF. Finally, mitochondria itself could be secret-
ing an unidentified extracellular signal, called by Durieux et al.
a “mitokine”, which is then transmitted to remotely located cells
[82].
3) Nerves (Repair and Pain): Some clinical studies have
demonstrated the efficacy of laser-induced analgesia [121],
[122], usually with a low power red or near-infrared laser, and
it seems that the pain reduction is due to a conduction block of
central and peripheral nerve fibers and to the release of endor-
phins. In this field, for instance, Chan and co-workers used a
Nd:YAG pulsed laser (1064 nm) with average power 1.2 W and
power density 0.3-0.45 J cm2in a randomized, double-blind
clinical trial, and demonstrated the efficacy of this treatment on
pulpal analgesia of premolar teeth [123].
Analgesia mediated by low level light therapy is due to various
effects, such as light absorption by mitochondrial chromophores
(mainly Cox) biomodulation, vasodilation, stimulation of cell
division, release of NO, increase in cortisol levels and protein
synthesis, increase in intracellular calcium concentration and
increased activity of the antioxidant enzyme superoxide dis-
mutase. Serra and Ashmawi investigated recently if serotonin
played a role in PBM-induced analgesia, but their results indi-
cated that this effect is mediated by peripheral opioid receptors,
but not by peripheral serotoninergic receptors [124].
Low-level light therapy can be used for inhibition of pain and
for pathological conditions associated with the nervous system.
In 2011, Yan et al. postulated that PBM could suppress afferent
fiber signaling as well as modulate synaptic transmission to
dorsal horn neurons, including inhibition of substance P, and this
can lead to long-term pain depression [125]. PBM exerts potent
anti-inflammatory effects in the peripheral nervous system, can
reduce myocardial infarction, promotes functional recovery and
regeneration of peripheral nerves after injury, and can improve
neurological deficits after stroke and TBI [82].
Light with irradiance higher than 300 mW cm2, when ab-
sorbed by nociceptors, can inhibit Aδand C pain fibers, slowing
of conduction velocity, reducing of the compound action poten-
tial amplitude, and suppression of neurogenicin inflammation.
In case of PBM, the light can block anterograde transport of
ATP-rich mitochondria in dorsal root ganglion neurons. This
inhibition is completely reversible within 48 hours, and leads to
the formation of varicosities, which are usually associated with
the disruption of microtubules (interruption of fast axonal flow
can reduce ATP availability, which is necessary for the polymer-
ization of microtubules and for the maintenance of the resting
potential) [83].
4) Healing (Bones, Tendons, Wounds): Regarding bones,
low power laser irradiation is not believed to affect osteosyn-
thesis, but it is likely that it creates environmental conditions
that accelerate bone healing. PBM stimulates proliferation and
differentiation of osteoblasts in vivo and in vitro, leading to an
increased bone formation, accompanied by an increase in the
7000417 IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 22, NO. 3, MAY/JUNE 2016
activity of alkaline phosphatase and in osteocalcin expression.
This indicates that laser irradiation can directly stimulate bone
formation and, according to Fujimoto et al., this effect can be at-
tributed to an increased expression of insulin-like growth factor
(IGF), although other differentiation factors might be involved
as well, such as BMPs. BMPs-2, -4, -6 and -7 are members of
the TGF-βsuperfamily, and potent promoters of osteoblastic
differentiation and of bone formation (promoting the change of
mesenchymal cells into chondroblasts and osteoblasts) [126].
According to Fujimoto, BMP-2 might be most involved in the
effects of PBM on bone. PBM stimulated mineralization in vitro
via increased gene and protein expression of BMPs and Runx-
2, as well as differentiation of osteoblasts into MC3T3-E1cells.
Since BMPs are one of the most important and potent bone-
inductive mediators and are expressed in skeletal tissues, it is
possible that the bone nodules formed after PBM are mediated
in part by BMP-2 expression [126].
The balance between oxidants and antioxidants is directly
related to the time and quality of the wound healing process
[127]. This process can be divided in four overlapping phases:
hemostasis, inflammation, proliferation and remodeling or res-
olution. Hemostasis is initiated as soon as the blood vessels are
damaged, and consists on the adherence of platelets to the extra-
cellular matrix and further releasing of growth factors (mostly
platelet-derived growth factor, PDGF and TGF-β), culminating
in the production of thrombin which acts on fibrinogen to pro-
duce a fibrin clot. Thrombin also acts as a chemotactic agent
and proliferating agent on monocytes, keratinocytes, fibroblasts
and endothelial cells, therefore a defective thrombin activity can
lead to a delay in the wound healing process. Hoffman reported
that PBM could be beneficial in promoting healing when there
is a defect in the hemostasis process [128].
5) Hair: Different mechanisms have been proposed to ex-
plain the reason for the first light-mediated effect observed by
Mester in 1968 (hypertrichosis in mice [2]) but now widely
used clinically to restore hair growth in adult humans [129].
Some researchers have hypothesized that this effect was due to
polycystic ovarian syndrome present in 5 out of 49 female pa-
tients under laser treatment for facial hirsutism, others suggested
that even if the heat generated by PBM was not able to ablate
cells from the hair follicle, the small amount of heat supposedly
produced could induce follicular stem cells to proliferate and
differentiate, due to the increased level of HSP. Another possi-
bility relies on the release of certain factors that could affect the
cell cycle and induce angiogenesis [129]. The exact mechanism
still needs clarifying, but the effects of PBM on hair growth are
already well described.
Hair growth is divided basically in three phases: anagen, cata-
gen and telogen. The anagen is the growth phase and can last
from 2 to 6 years. Catagen phase lasts from 1 to 2 weeks and
consists of club hair transitions upwards toward the skin pore,
while the dermal papilla separates from the follicle. In the tel-
ogen phase, the dermal papillae fully separate from the hair
follicle. It lasts from 5 to 6 weeks, until the papillae move up-
ward to meet the hair follicles again and the hair matrix begins
to form new hair, returning to the anagen phase. It has been
observed that PBM is able to stimulate telogen hair follicles
to enter the anagen phase, as well as to prolong the duration
of the anagen phase itself. PBM is also capable of increasing
the rate of proliferation of anagen hair follicles and to prevent
premature catagen phase entry. This could be due to induced
protein synthesis by the transcription factors activated by PBM,
followed by cell migration and proliferation, alteration in cy-
tokines levels, growth factors and inflammatory mediators. NO
is also augmented in LLL treated tissues, usually dissociated
from Cox, and since it is a well known vasodilator, it is likely
that there is a vasodilation effect on hair follicles after PBM that
could help hair growth. Some inflammatory mediators also have
their expression inhibited by PBM (such as IFN-γ, IL-1a, IL-
1b, TNF and Fas-antigen) and, considering that inflammation is
highly disruptive for hair follicles, the anti-inflammatory effect
of PBM could be useful in the treatment of hair conditions such
as alopecia areata [129].
G. High Fluence Low Power Laser Irradiation (HF-LPLI)
Fluence, according to the International System of Units, is
the energy density integrated over the unit surface of a sphere.
Just like PBM using low fluences of light, high-fluence low-
power laser irradiation (HF-LPLI) stimulates mitochondrial
chromophores, but this time it overstimulates them, which in
turn activates the mitochondrial apoptosis pathway, altering the
cell cycle, inhibiting cell proliferation and even causing cell
death. HF-LPLI (usually fluences above 80 J cm2) induces
apoptosis by activating caspase-3, and mitochondrial perme-
ability transition after HF-LPLI is the main mechanism of mi-
tochondrial injury. In 2010, Sun et al. reported that signal trans-
ducer and activator of transcription 3 (Stat3) was involved in
HF-LPLI-induced apoptosis in vitro, and this effect is time- and
dose-dependent. Steroid receptor coactivator (Src) seems to be
the main upstream kinase of Stat3 activation, and the increased
ROS generation plays a key role in this process [130].
Recently, Wu et al. found that HF-LPLI, using light at 633 nm
and 120 J cm2, could ablate tumors via activation of mitochon-
drial apoptotic pathway after ROS generation. The evidence is
based on the inactivation of caspase-8, activation of caspase-
9 and by the release of cytochrome C. When this high dose
is used, light inactivates Cox (instead of activating Cox), in-
ducing a superoxide burst in the electron transport chain and,
finally, produces oxidative damage against cancer cells [29].
Chu and co-workers already observed that PBM could induce a
mitochondrial permeability pore transition when higher levels
of ROS are produced. As a consequence, the decrease of mi-
tochondrial transmembrane potential causes the permeabiliza-
tion of the mitochondrial outer membrane and, subsequently,
the release of cytochrome c and caspase cascade reaction
[131].
Cho also observed the interference that a protein, called sur-
vivin, could affect the outcomes of HF-LPLI. Light treatment
can activate survivin by inducing an increase in its phosphory-
lation levels. The activated survivin is able to inhibit the per-
meabilization of the mitochondrial outer membrane, and there-
fore prevents the release of cytochrome c, the activation of Bax
and caspase-9. Cho then concluded that survivin mediates self
DE FREITAS AND HAMBLIN: PROPOSED MECHANISMS OF PHOTOBIOMODULATION OR LOW-LEVEL LIGHT THERAPY 7000417
protection of tumor cells against HF-LPLI-induced apoptosis,
through ROS/cdc25c/CDK1 signaling pathway [131].
V. CONCLUSION
Low levels of red/NIR light can interact with cells, leading
to changes at the molecular, cellular and tissue levels. Each tis-
sue, however, can respond to this light-interaction differently,
although it is well known that the photons, especially in the red
or NIR, are predominantly absorbed in the mitochondria [132].
Therefore, it is likely that even the diverse results observed with
PBM share the basic mechanism of action. What happens after
the photon absorption is yet to be fully described, since many
signaling pathways seem to be activated. It seems that the effects
of PBM are due to an increase in the oxidative metabolism in
the mitochondria [133]. Different outcomes can occur depend-
ing on the cell type, i.e. cancer cells that tend to proliferate when
PBM is delivered [88]. In this review we have not discussed the
response of cells and tissues to wavelengths longer than NIR,
namely far IR radiation (FIR) (3 to 50 μm). At these wave-
lengths water molecules are the only credible chromophores,
and the concept of structured water layers that build up on bio-
logical lipid bilayer membranes has been introduced to explain
the selective absorption [134]. Nevertheless FIR therapy has
significant medical benefits that are somewhat similar to those
of PBM [135], and it is possible that activation of light/heat
sensitive ion channels could be the missing connection between
the two approaches.
As we have shown, PBM can regulate many biological pro-
cesses, such as cell viability, cell proliferation and apoptosis,
and these processes are dependent on molecules like protein
kinase c (PKC), protein kinase B (Akt/PKB), Src tyrosine ki-
nases and interleukin-8/1a (IL-8/1a). The effects of light on cell
proliferation can be stimulatory at low fluences (which is use-
ful in wound healing, for instance), but could be inhibitory at
higher light doses (which could be useful in certain types of scar
formation such as hypertrophic scars and keloids) [131].
The applications of PBM are broad. Four clinical targets,
however, are the most common: shining light on injured sites to
promote healing, remodeling and/or to reduce inflammation; on
nerves to induce analgesia; on lymph nodes in order to reduce
edema and inflammation; and on trigger points (a single one
of as many as 15 points) to promote muscle relaxation and to
reduce tenderness. Since it is non invasive, PBM is very useful
for patients who are needle phobic or for those who cannot
tolerate therapies with non-steroidal anti-inflammatory drugs
[83].
The positive outcomes depend on the parameters used on the
treatment. The anti-inflammatory effect of light in low intensity
was reported on patients with arthritis, acrodermatitis continua,
sensitive and erythematous skin, for instance [136]. With the
same basic mechanism of action, which is the light absorption
by mitochondrial chromophores, mainly Cox, the consequences
of PBM are various, depending on the parameters used, on the
signaling pathways that are activated and on the treated tissue. In
order to apply PBM in clinical procedures, the clinicians should
be aware of the correct parameters and the consequences for
each tissue to be treated. More studies have to be performed in
order to fill the gaps that still linger in the basic mechanisms
underlying LLLT and PBM.
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Lucas Freitas de Freitas received the Graduate de-
gree from State University of Londrina, Londrina,
Brazil, in 2009 and the Ph.D degree in bioengineer-
ings from University of S˜
ao Paulo - Brazil in 2016.
Part of the Ph.D. research was performed under the
supervision of Dr. M. Hamblin from the Wellman
Center for Photomedicine - Harvard Medical School
(2014–2015). He is a Biomedical Professional. He
has publications in the biomedical field, especially
on light interaction with microorganisms and with
the human body, cancer therapies and diagnostics,
and oxidative stress.
Michael R Hamblin received the Ph.D. degree from
Trent University, Nottingham, England. He is a is
a Principal Investigator at the Wellman Center for
Photomedicine at Massachusetts General Hospital,
an Associate Professor of Dermatology at Harvard
Medical School, and is a member of the affiliated
faculty of the Harvard-MIT Division of Health Sci-
ence and Technology. He was trained as a Synthetic
Organic Chemist. His research interests include pho-
todynamic therapy for infections, cancer, and stimu-
lation of the immune system, and in low-level light
therapy for wound healing, arthritis, traumatic brain injury, neurodegenerative
diseases and psychiatric disorders. He directs a laboratory of around a dozen
post-doctoral fellows, visiting scientists, and graduate students. His research
program is supported by NIH, CDMRP, USAFOSR, and CIMIT among other
funding agencies. He has published more than 320 peer-reviewed articles, over
150 conference proceedings, book chapters, and International abstracts, and
holds 10 patents. He is Associate Editor for 7 journals, on the editorial board of
a further 30 journals and serves on NIH Study Sections. For the past 11 years,
he has chaired an annual conference at SPIE Photonics West entitled “Mecha-
nisms for low level light therapy” and he has edited the 11 proceedings volumes
together with four other major textbooks on PDT and photomedicine. He has
several other book projects in progress at various stages of completion. In 2011,
he was honored by election as a Fellow of SPIE. He is a Visiting Professor at
universities in China, South Africa, and Northern Ireland.
... Red light (RL) phototherapy may also be a beneficial adjunctive melanoma therapy by inhibiting tumor growth and augmenting anti-tumor immune activity. RL may non-thermally alter cellular biology via a process termed photobiomodulation (9). RL absorption by cytochrome c oxidase, a photoreceptive chromophore, excites electrons, activates the electron transport chain, and generates second messengers such as reactive oxygen species (ROS) (9,10). ...
... RL may non-thermally alter cellular biology via a process termed photobiomodulation (9). RL absorption by cytochrome c oxidase, a photoreceptive chromophore, excites electrons, activates the electron transport chain, and generates second messengers such as reactive oxygen species (ROS) (9,10). Cancers, including melanoma, have dysregulated ROS homeostasis and may be particularly sensitive to oxidative stress (9,11). ...
... RL absorption by cytochrome c oxidase, a photoreceptive chromophore, excites electrons, activates the electron transport chain, and generates second messengers such as reactive oxygen species (ROS) (9,10). Cancers, including melanoma, have dysregulated ROS homeostasis and may be particularly sensitive to oxidative stress (9,11). Increasing intracellular ROS in cancer cells may lead to cell death or inhibition of growth and metastasis (12)(13)(14). ...
Article
Full-text available
Background Total annual cancer rates have decreased due to improved treatment and prevention. However, the incidence of melanoma is rising, and not all patients respond to immune and targeted approaches. Therefore, we sought to determine the efficacy of red light (RL) phototherapy in preclinical models of melanoma. Methods Melanoma cells (A375, B16F10, MNT-1) were irradiated with RL. Melanoma proliferation, apoptosis, oxidative stress, and p53 phosphorylation were measured in vitro . In C57BL/6 mice, phototherapy safety, B16F10 tumor growth, and immunocyte infiltration were assessed following RL. Results In vitro , 640 J/cm ² RL decreased cellular proliferation without increasing apoptosis, while 1280 J/cm ² increased apoptosis. RL increased intracellular reactive oxygen species generation and p53 phosphorylation. In animal models, 2560 J/cm ² RL significantly prevented melanoma growth and increased the expression of CD103+ dendritic cells. 1280 and 1920 J/cm ² RL decreased tumor volume, but not significantly. RL did not cause skin inflammation or erythema in normal skin. Conclusion RL represents a potentially safe and effective melanoma therapeutic. RL prevented tumor growth and increased the expression of immune markers, such as CD103, that are associated with favorable melanoma outcomes. Further research is needed to determine the optimal clinical treatment regimen for melanoma using RL.
... In the last decades, light has been increasingly applied in research, survey, treatment, and therapy of biological objects (considering here the term light for near-ultraviolet (UV), visible, and nearinfrared (NIR) irradiation); it is named as low-level laser therapy (LLLT) and more generally as photobiomodulation (PBM) or photobiomodulation therapy (PBMT) (1,2). There are few known independent light-sensitive cellular pathways as the activation of ion channels on cell membrane and endoplasmic reticulum (3,4), though, light principally interacts with mitochondria, which is the main source of cellular reactive oxygen species (ROS) (5)(6)(7). Light absorption by endogenous chromophores and photoacceptor causes changes in the mitochondrial ultrastructure, resulting in an increase of the mitochondrial membrane potential (MtMP) and triggering photochemical reactions by several intracellular signaling pathways (5). ...
... To date, the most discussed photoacceptor is cytochrome c oxidase (CCO) (6,13). CCO absorbs red/NIR light within wavelengths ranging between 600-1000 nm, resulting in the activation of metabolic process as well as migration and proliferation of various cell types: neurons (650-660/808 nm at 3-90 J/cm 2 (3,4,14), fibroblasts (660 nm at 5 J/cm 2 (15), 660 nm at 3-8 J/cm 2 (16), 809 nm at 1.96-7.84 ...
Article
Full-text available
The effect of UV/visible/NIR light (380/450/530/650/808/1064 nm) on ROS generation, mitochondrial activity, and viability is experimentally compared in human neuroblastoma cancer cells. The absorption of photons by mitochondrial photoacceptors in Complexes I, III, IV is in‐detail investigated by sequential blocking with selective pharmaceutical blockers. Complex I absorbs UV/blue light by heme P450, resulting in a very high rate (14 times) of ROS generation leading to cell death. Complex III absorbs green light, by cytochromes b, c1 and c, and possesses less ability for ROS production (7 times), so that only irradiation lower than 10 mW/cm2 cause an increase in cell viability. Complex IV is well‐known as the primary photoacceptor for red/NIR light. Light of 650/808 nm at 10–100 mW/cm2 generates a physiological ROS level about 20% of a basal concentration, which enhance mitochondrial activity and cell survival. While, 1064 nm light does not show any distinguished effects. Further, ROS generation induced by low‐intensity red/NIR light is compared in neurons, immune and cancer cells. Red light seems to more rapidly stimulate ROS production, mitochondrial activity, and cell survival than 808 nm. At the same time, different cell lines demonstrate slightly various rates of ROS generation, peculiar for their cellular physiology.
... We designed experiments to reveal biological changes in hADSCs promoted by green OLED irradiation and their underlying mechanisms. It has been suggested that CCO plays a major role in cellular responses of stem cell to PBM [49]. OPNs besides CCO are also related to responses to PBM [18]. ...
... Although we did not observe significant difference in intracellular Ca 2+ concentration, both cellular ROS assay and H2DCFDA staining showed increases in ROS level and gene expression levels of PLC-β and IP3R known to mediate ER Ca 2+ release [50] were elevated in green OLED-treated group of hADSCs. These results indicate that green OLED irradiation does not follow the molecular mechanism of TRP channels or the CCO pathway, the proposed mechanisms in PBM when using red or near-infrared light in the range of 600-700 nm and 780-1100 nm [49]. As shown in our results, green OLED treatment had no obvious cytotoxic effect on hADSCs despite generation of ROS. ...
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Background Human adipose-derived stem cells (hADSCs) have been widely used for regenerative medicine because of their therapeutic efficacy and differentiation capacity. However, there are still limitations to use them intactly due to some difficulties such as poor cell engraftment and viability after cell transplantation. Therefore, techniques such as photobiomodulation (PBM) are required to overcome these limitations. This study probed improved preclinical efficacy of irradiated hADSCs and its underlying molecular mechanism. Methods hADSCs were irradiated with green organic light-emitting diodes (OLEDs). Treated cells were analyzed for mechanism identification and tissue regeneration ability verification. Expression levels of genes and proteins associated with photoreceptor, cell proliferation, migration, adhesion, and wound healing were evaluated by performing multiple assays and immunostaining. Excision wound models were employed to test in vivo therapeutic effects. Results In vitro assessments showed that Opsin3 (OPN3) and OPN4 are both expressed in hADSCs. However, only OPN4 was stimulated by green OLED irradiation. Cell proliferation, migration, adhesion, and growth factor expression in treated hADSCs were enhanced compared to control group. Conditioned medium containing paracrine factors secreted from irradiated hADSCs increased proliferation of human dermal fibroblasts and normal human epidermal keratinocytes. Irradiated hADSCs exerted better wound healing efficacy in vivo than hADSCs without OLED irradiation. Conclusions Our study introduces an intracellular mechanism of PBM in hADSCs. Our results revealed that photoreceptor OPN4 known to activate G q -protein and consequently lead to reactive oxygen species production responded to OLED irradiation with a wavelength peak of 532 nm. In conclusion, green OLED irradiation can promote wound healing capability of hADSCs, suggesting that green OLED has potential preclinical applications.
... The PBM device delivers red and near infrared (NIR) light to selected areas of the body, stimulating mitochondrial activity 3 . The mechanisms include the release of nitric oxide (NO) in the mitochondria 4 3 which has been shown to inhibit the replication of exposed coronavirus 5 6 7 8 9 and support endothelial function 10 11 , beneficial to patients with acute respiratory distress syndrome (ARDS) and impaired pulmonary function 12 13 14 , which are features of acute COVID-19 15 16 17 . ...
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BACKGROUND There is need for non-pharmaceutical treatments for COVID-19. A home-use photobiomodulation (PBM) device was tested as Treatment in a randomized clinical trial. METHODS 294 patients were randomized with equal allocation to Treatment or Standard of Care (Control). 199 qualified for efficacy analyses. The Treatment group self-treated for 20 minutes twice daily, for the first 5 days, and subsequently once daily for 30 days. A validated respiratory questionnaire was used, and patients were monitored remotely. The primary endpoint was the time-to-recovery (3 consecutive days of no sickness) for general sickness. The Kaplan-Meier method and the Cox Proportional Hazards model were primary methods of analyses. RESULTS Treatment patients with collective 0-12 days of symptoms, at moderate-to-severe level on Day 1 of Treatment, did not recover significantly faster than Control. However, for patients with 0-7 days of symptoms there was a significant mean difference of 3 days: Treatment, 18 days (95% CI, 13-20) vs. Control, 21 days (95% CI, 15-28), P=0.050. The Treatment:Control hazard ratio at 1.495 (95% CI, 0.996-2.243), P=0.054 exceeded the pre-trial target of 1.44. Treated patients exceeding 7 days symptoms duration were more tired and had lower energy. None of the patients in the Treatment group suffered death or hospitalization while the Control group had 1 death and 3 severe adverse events requiring hospitalization. CONCLUSIONS Patients with up to 7 days of symptoms at moderate-to-severe levels on first day of Treatment can expect faster recovery for general sickness and several respiratory symptoms. (Funded by Vielight Inc.; ClinicalTrials.gov number, NCT04418505 .)
... This process can increase the generation of ATP and change the levels of ROS (61,62). More research is necessary to confirm our hypothesis and further explore the antioxidant mechanism of IPL. ...
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Low-level laser therapy (LLLT) continues to receive much attention in many clinical fields. Also, LLLT has been used to enhance the proliferation of various cell lines, including stem cells. This study investigated the effect of LLLT on human adipose-derived stem cells (ADSCs) through in vitro and in vivo studies. Low-level laser irradiation of cultured ADSCs was performed using a 830 nm Ga-Al-As (gallium-aluminum-arsenide) laser. Then, proliferation of ADSCs was quantified by a cell counting kit-8. In the in vivo study, irradiated ADSCs or non-irradiated ADSCs were transplanted, and then, low-level laser irradiation of each rat was performed as per the protocol. Cell viability was quantified by immunofluorescent staining using the human mitochondria antibody. In the in vitro study, the laser-irradiated groups showed an increase in absorbance compared to the control group. Also, in the in vivo study, there was a significant increase in the number of human ADSCs in the laser-irradiated groups compared to the control group (p < 0.001). Our study showed that LLLT could enhance the proliferation and viability of ADSCs. The ADSCs enhanced by LLLT could be applied in various clinical fields. With the use of LLLT, the proliferation and viability of various cells can be enhanced, besides ADSCs. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .
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Calcium (Ca2+) release from intracellular stores controls numerous cellular processes, including cardiac and skeletal muscle contraction, synaptic transmission and metabolism. The ryanodine receptors (RyRs) and inositol 1,4,5--trisphosphate receptors (IP3Rs) are the majorCa2+ release channels (CRCs) on the endo/sarcoplasmic reticulum(ER/SR).RyR1 and RyR2 are the key isoforms in skeletal and cardiac muscle and are essential role in excitation--contraction(E--C) coupling. IP3R1 and IP3R2 are required for muscle and neuronal function. RyRs and IP3Rs comprise macromolecular signaling complexes that include modulatory proteins which regulate channel activity in response to extracellular signals resulting in intracellular Ca2+ release. This review focuses on the roles of CRCs in heart, skeletal muscle, brain, and aging.
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The aim of this study was to investigate the effects of different low-level laser therapies (LLLTs) of various wavelengths and energies on normal cultured human melanocytes. Various studies have shown the effects of LLLs on various types of cultured cells. Presently, little is known about the biological effects of LLLTs on melanocytes. Melanocytes were exposed to LLLT at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 J/cm(2) using a blue (457 nm), red (635 nm), or ultraviolet (UV) (355 nm) laser. Melanocyte viability, proliferation, and migration were monitored at 72 h after irradiation. The blue (P < 0.001) and red (P < 0.001 and P < 0.01) lasers significantly enhanced viability at 0.5 to 2.0 J/cm(2), whereas the UV laser (P < 0.001) could significantly enhance viability only at 0.5 and 1.0 J/cm(2) compared with controls. The blue and red lasers also significantly enhanced the proliferation of the melanocytes at 0.5 to 2.0 J/cm(2) (P < 0.001), and the UV laser significantly enhanced proliferation at 0.5 to 1.5 J/cm(2) (P < 0.001 and P < 0.01) compared with controls. The blue laser significantly enhanced melanocyte migration at 0.5 to 4.0 J/cm(2) (P < 0.001 to P < 0.05), but the red (P < 0.001 and P < 0.01) and UV (P < 0.001 to P < 0.05) lasers could significantly enhance such migration at 0.5 to 1.0 J/cm(2) and 0.5 to 2.0 J/cm(2), respectively, compared with controls. LLLT at low energy densities is able to significantly increase melanocyte viability, proliferation, and migration in vitro, and at higher energy densities, it gives non-stimulatory results. Additionally, the blue laser was the best among the three lasers. These findings might have potential application in vitiligo treatment in future.
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Macrophages are a source of many important mediators of wound repair. Cells of an established macrophage-cell line (U-937) were exposed in vitro to an 820 nm light source which was both coherent and polarized. the power densities used being either 400 mW/cm2 or 800 mW/cm2. The irradiation times were such that the energy densities to which the cells were exposed were 2.4 and 7.2J/cm2 for both probes. Twelve hours after exposure the macrophage-conditioned medium was removed and placed on 3T3 fibroblast monolayers. Fibroblast proliferation was assessed over a four-day period. By four days after the addition of medium conditioned by macrophages exposed to an energy density of 2.4 J/cm2, there was a statistically significant difference in fibroblast number between the 400 mW/cm2- and 800 mW/cm2 treatments, 800mW/cm2 producing greater cell proliferation. However. there was no significant difference between the effects of sham irradiation and 400 mW/cm2. In contrast, after the addition of medium conditioned by macrophage exposed to an energy density of 7.2 J/cm2, 400 mW/cm2 treatment produced a significantly greater increase in fibroblast number than sham irradiation. There was no significant difference in cell number between the sham irradiated and 800 mW/cm2 irradiated samples, although there was a significant difference between the 400 mW/cm2 and the 800 mW/cm2. 400 mW/cm2 producing greater cell proliferation.
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The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Cao(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (Gq/11, Gi/o, G12/13, and Gs) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular elements (e.g., the cytoskeleton) to facilitate the interaction of the receptor with its signaling pathways. Regulatory mechanisms are still evolving to understand how defects in trafficking of CaSR contribute to pathology in disorders of calcium homeostasis. © 2015 Published by Elsevier Inc. All rights reserved.
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Calcium is thought to play an important role in regulating mitochondrial function. Evidence suggests that an increase in mitochondrial calcium can augment ATP production by altering the activity of calcium-sensitive mitochondrial matrix enzymes. In contrast, the entry of large amounts of mitochondrial calcium in the setting of ischemia-reperfusion injury is thought to be a critical event in triggering cellular necrosis. For many decades, the details of how calcium entered the mitochondria remained a biological mystery. In the past few years, significant progress has been made in identifying the molecular components of the mitochondrial calcium uniporter complex. Here, we review how calcium enters and leaves the mitochondria, the growing insight into the topology, stoichiometry and function of the uniporter complex, and the early lessons learned from some initial mouse models that genetically perturb mitochondrial calcium homeostasis. © 2015 American Heart Association, Inc.