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Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7441
Samith Ahmed1, Gregory Bewsh2, Shankaranarayana Bhat3, Ramesh Babu4
Samith Ahmed, Gregory Bewsh, Shankaranarayana Bhat, Ramesh Babu Low level laser therapy: healing at the
speed of light. Journal of Evolution of Medical and Dental Sciences 2013; Vol2, Issue 39, September 30; Page:
KEYWORDS: Laser, ATP, Mitochondria, Wound.
Therapy (LLLT) is a rapidly growing modality used in rehabilitation and physical therapy. A number
of safe and beneficial therapeutic effects of LLLT have been reported in numerous clinical
conditions; however, despite many reports of positive findings from experiments conducted in-vitro,
in animal models and in randomized controlled clinical trials worldwide, the use of this scientifically
grounded, non-invasive, anti-inflammatory and regulatory modality has yet to find mainstream
adoption by medical doctors. The aim of this review is six fold:
1) Introduce the unfamiliar reader, with some medical background, to the contemporary concept
of LLLT and its pathophysiological significance,
2) Review the role of the mitochondrial pathway in the mechanisms of LLLT,
3) Provide necessary practical guidance based on personal scientific and clinical experience,
4) Assist manufacturers in their research and development,
5) Help health care practitioners choose and use an adequate light therapy device
6) Outline the prospects of LLLT as an avenue to treat chronic inflammation and pain and to aid
in an effective clinical practice.
THE STATE OF THE ART: Low Level Laser Therapy (LLLT) is the drug-free, non-invasive and safe
(FDA recognised) clinical application of light (usually produced by a low to mid power coherent
lasers or non-coherent light emitting diodes (LEDs) in the range of 1mW 500mW) to a patient to
promote tissue regeneration, reduce inflammation and relieve pain. The light is typically of narrow
spectral range (1 to 40 nm bandwidth) in the visible (red) 600 to 700 nm or near infrared (NIR)
spectrum (700nm to 950nm), achieving an average power density (irradiance) between. 001 to 5
Light irradiation is typically applied onto the affected area (the areas where the treatment is
needed) or pain projected area or suggested acupuncture points for a few seconds to several
minutes, daily, or multiple times per week, for a treatment period of up to 8 to 10 weeks depending
on chronicity. Thus in treatment frequency and duration LLLT does not differ from other modalities,
such as thermal heating blankets⁽¹⁾, electrostimulation⁽²⁾ or ultrasound⁽³⁾; however, as we shall see
dramatically different in cellular mechanism and efficacy.
LLLT is not considered an ablative or destructive procedure on the tissue or cellular level
and employs biomodulation via photophysical and potential photochemical mechanisms,
comparable to photosynthesis in plants; whereby, the absorbed light is able to initiate a cascade of
molecular and functional regulatory changes.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7442
The reason why the technique is generally referred to as Low Level Laser Therapy (LLLT) is
that the optimal level of energy density delivered to the tissues is low when compared to other
forms of laser therapy such as the densities required for ablation, cutting, and thermally coagulating
tissue. In general, the power densities used for LLLT are lower than those needed to produce heating
of tissue (i. e. less than 500 mWcm-2, depending on wavelength and tissue type), and the total
energy delivered to the treated area is commonly below 1J cm- ². A common reference is the
“Guidelines for Skin Exposure to Laser Light” in the International Standards Manual (IEC-825) which
considers exposure below 200mW cm-2 as a safe exposure (4) in the visible range, escalating to
approximately 500 mW/cm² in the near infrared range.
The use of low levels of visible or near infrared light (LLLT) (5), for reduction of
inflammation, elimination of pain (6), healing of wounds, repair to nerves injuries and (7)
prevention of tissue damage by reducing cellular apoptosis (for example, due to ischemia and
reperfusion injury), has been known for fifty years since shortly after the invention of lasers in the
early 60s.
Despite many reports of positive findings from experiments conducted in-vitro, in-vivo
(animal and human models) and in randomized controlled clinical trials, the mechanisms of action of
LLLT in biological tissue remains not fully elucidated. This likely is due to two main reasons: firstly
the complexity in interpretation of scientific and clinical data generated in different labs and clinical
settings and secondly variability in the use of light sources (medical devices) and treatment
protocols utilized including, illumination parameters (such as: wavelength, fluence, power density,
pulse structure, etc.) and the treatment schedule. These factors lead to many machinations of factors
that make side to side comparisons difficult.
As a result, the original field of Low Level Laser (coherent-monochromatic light) Therapy
(LLLT) or Low Level Light Therapy (LLLT) subject has now expanded to include photo
biomodulation and photobiostimulation using non-laser (non-coherent light) instruments.
These variation in light sources utilized have led to an increase in the number of negative
studies and created some controversy despite the overwhelming number of positive clinical
results⁽¹⁾. It is noteworthy that many studies have been conducted without proper scientific
methodology, as all the characteristics of the light emitted by lasers or LEDs must be specified if a
paper is to be useful. A requirement for a good phototherapy study, using low intensity radiation in
the visible or near-infrared region, whether from a laser, an LED, or a filtered incandescent lamp is
to specify everything about the light source, i. e., wavelength(s), power, dose, area of exposure, time,
etc. There are numerous published experimental and clinical studies that were conducted with good
scientific methodology, but unfortunately they did not describe the light source⁽⁸⁾; therefore, these
studies cannot be repeated or extended by another author. Such publications are useless for
progressing the field of science of LLLT.
In recent years, major advances have been made in understanding the mechanisms that
operate at the cellular and tissue levels during LLLT. Mitochondria are thought to be the main site
for the initial effects of light. The discovery and universal acceptance of this phenomenon is of
particular significance to Laser manufactures, because both mitochondria membrane lipids and the
large mitochondrion transmembrane protein complex (cytochrome c oxidase) have absorption
spectra and action spectra peaks in the red and near infrared regions of the electromagnetic
spectrum matching the emission spectra of the 660 nm and 905 nm, dual wavelengths of Theralase’s
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7443
LLLT devices. Thus a photochemical activation of pathways involving these transmembrane protein
complexes as well as potential subcellular photothermal effects is possible (6).
New discoveries in the last decade significantly altered our view on mitochondria. They are
no longer viewed as energy-making cellular compartments but rather individual cells within- the-
cell. In particular, it has been suggested that many important cellular mechanisms involving specific
enzymes and ion channels, such as nitric oxide synthase (NOS), ATP-dependent K+ (KATP) channels,
and poly-(APD-ribose) polymerase (PARP), have a distinct, mitochondrial variant. For example, the
intriguing possibility that mitochondria are significant sources of nitric oxide (NO) via a unique
mitochondrial NOS variant has attracted intense interest among research groups because of the
potential for NO to affect functioning of the electron transport chain (8).
It has been shown that LLLT mediated effect may employ inducible nitric oxide synthase
(iNOS) to potentially activate production of nitric oxide (NO) in mitochondria (6). The discovery of
this phenomenon supports a concept that NO and its derivatives (reactive nitrogen species) have
multiple effects on mitochondria that may impact a cell’s physiology and the cell’s cycle (7).
It was shown that inducible in mitochondria nitric oxide (iNO) inhibits mitochondrial
respiration via: (A) an acute and reversible inhibition of cytochrome c oxidase by NO in competition
with O2, and (B) irreversible inhibition of multiple sites by reactive nitrogen species. iNO stimulates
reactive oxygen and nitrogen species production from mitochondria via respiratory inhibition,
reaction with ubiquinol and reaction with O2 in the mitochondrial membranes. (9) Oxidants/free
radicals may also confer physiological functions and cellular signaling processes (10) due to iNO.
According to Brown et al., mitochondria may produce and consume NO and NO stimulates
mitochondrial biogenesis, apparently via upregulation of nucleotides like ATP and transcriptional
factors like nuclear factor kappa B (NF kb) (9).
Therefore, it can be suggested that the super pulsed 905nm LLLT-induced NO can reprogram
cellular function, mainly via oxidative stress and changes of mitochondrial temperature gradient due
to process known as selective photo thermolysis and consequently initiate a cascade of local and
systemic therapeutic signaling (6).
These signal transduction pathways in turn may lead to increased cell activation and traffic,
modulation of regulatory cytokines, growth factors and inflammatory mediators, and expression of
protective (anti-apoptotic) proteins (11; 12).
The results of these molecular and cellular changes in animals and patients integrate such
benefits as increased: healing in chronic wounds, improvements in sports injuries and carpal tunnel
syndrome, pain reduction in arthritis and neuropathies, amelioration of damage after heart attacks,
stroke, nerve injury and alleviation of chronic inflammation and toxicity (13).
The LLLT-induced NO may explain 1) generation of reactive oxygen species (ROS), induction
of transcription factors and increased ATP production (in cells) 2) modulation of immune responses,
reduction and prevention of apoptosis, regulation of circulation and angiogenesis (in tissues), and
suppression of local and systemic inflammation and pain. LLLT-induced ROS, iNO and ATP mediated
signaling cascades are well documented in the peer-reviewed literature (14; 15; 16; 17; 18).
More evidence now suggests that LLLT is a rapidly growing modality used in physical
therapy, chiropractic and sport medicine and increasingly entering mainstream medicine. A full
spectrum of potential clinical targets that can be successfully treated by LLLT is continuing to grow
and at this point is not exhausted. LLLT is used to increase wound healing and tissue regeneration,
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7444
to relieve pain and inflammation, to prevent tissue death and to mitigate degeneration in many
neurological indications. We believe that further advances in elucidation of the LLLT’s mechanisms
will lead to greater acceptance of LLLT as the therapy of choice in the established market segments
and additionally as a first line or adjuvant therapy in other serious medical applications, currently
not being considered typical indications.
A SHORT HISTORY OF LLLT: Light therapy is one of the oldest therapeutic methods used by
humans; (historically as solar therapy by the Egyptians, Aztecs, Greeks and Romans and later as non-
ionizing UVB phototherapy of lupus vulgaris for which Niels Ryberg Finsen won the Nobel Prize for
Physiology in 1903 (14; 15).
The majority of the authors agreed that the era of LLLT starts in 1967, a few years after
Gordon Gould coined the acronym LASER (Light Amplification by Stimulated Emission of Radiation)
and described the essential elements for constructing a laser (1957) and 3 years after the Nobel
Prize for Physics (1964) was given to Nikolay Gennadiyevich Basov, Aleksandr Mikhailovich
Prokhorov and Charles Hard Townes for the invention of the MASER (microwave amplification by
stimulated emission of radiation) and the laser (16). The year 1967 is important in the history of
LLLT because of the ground breaking publication by Endre Mester and his colleagues (from
Semmelweis University, Budapest, Hungary). The authors uniquely demonstrated the therapeutic
benefit of monochromatic visible light and commenced the therapeutic laser field of science.
The investigators original expectation was to test if laser radiation might cause cancer in
mice. Mice were shaved and divided into control and treatment groups. The treatment group
received the light treatment with a low powered ruby laser (694 nm) while the control group did
not. The treated animals did not develop any malignancies and the treatment was safe; however, the
authors made a surprising observation about more rapid dorsal hair regrowth in the treated group
than the untreated group (17). Remarkably, the same study data can be considered as the first
disclosure of potential safety and efficacy of LLLT, particularly attributed to LLLT’s biostimulation
Since then, medical treatment with coherent light sources (lasers) or non coherent light
(LEDs) has passed through its experimental stage and is firmly rooted in clinical and scientific
documentation. Currently, low level laser (or laser therapy LLLT), also known as “cold laser”, “soft
laser”, “biostimulation” or photo biomodulation” is recognised by the FDA and practiced as part of
physical therapy in many parts of the world (18).
LLLT is showing promise in the treatment of a wide variety of medical conditions and has
been proven to be clinically safe and beneficial therapeutically in many tissues and organs (Figure
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7445
Currently, the knowledge of LLLT mechanisms continues to expand. Physicians are now
aware of LLLT’s potential to induce cellular and tissue effects through; for example, accelerated ATP
production and molecular signaling and that LLLT can be very effective in the treatment of various
serious clinical disorders.
In clinical use, however, it is often difficult to predict patient response to LLLT. It appears
that several key features, such as: the LLLT parameters and the treatment regimen (including,
irradiance [mW cm-2], radiant exposure [J cm-2], treatment regime (including the treatment mode,
pulsed vs. CW, and the treatment schedule), light attenuation [cm-2] in the tissues, etc.), cellular
pathophysiological status (including, reduction=oxidation (redox state), a disease localisation (depth
under the surface [mm]) tissue characteristics (including the tissue scattering parameters), the
character and the level of inflammatory process and variability of physiological and clinical
conditions in patients, can play a central role in determining sensitivity (and hence clinical efficacy)
to LLLT and may help to explain intra-individual variability in patient responsiveness to the
conducted therapy. Various cellular responses to visible and infrared radiation have been studied for
decades in the context of molecular mechanisms of low level non-coherent (non-laser) light and
laser phototherapy (19; 20; 21; 22).
It is generally accepted that the mitochondria are the initial site of light action in mammalian
cells, and cytochrome c oxidase (the terminal enzyme of the mitochondrial respiratory chain) is one
of the key responsible molecules (23) particular due to its broad absorption in the visible red and
NIR spectrum.
Figure 1: The diagram below represents the difference
in depth of penetration between therapeutic lasers.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7446
THE KEY MITOCHONDRIAL MECHANISMS OF LLLT: It is believed that in mammalian organisms
the mitochondrial changes taking place are playing the essential role in the mechanisms of LLLT and,
according to Hamblin and colleagues (2010), the response of cells to light is determined by the
mitochondrial number, their activity and membrane potential (24).
Mitochondria are the energy-transducing organelles of eukaryotic cells in which fuels that
drive cellular metabolism (i. e., carbohydrates and fats) are converted into adenosine triphosphate
(ATP) through the electron transport chain and the oxidative phosphorylation system (the
“respiratory chain”, Figure 2(29; 30).
Mitochondria are also involved in calcium buffering and the regulation of apoptosis (25).
They arose as intracellular symbiont in the evolutionary past, and can be traced to the prokaryote α-
proteobacterium (26). There are hundreds to thousands of mitochondria per cell (27), somewhat
dependent on the energy requirement of the individual cell. Structurally, mitochondria have four
compartments: the outer membrane, the inner membrane, the intermembrane space, and the matrix
(the region inside the inner membrane, see Figure 2, (30; 33; 34).
The respiratory chain is located on the inner mitochondrial membrane. It consists of five
multimeric protein complexes: reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase-
ubiquinone oxidoreductase (complex I), succinate dehydrogenase ubiquinone oxidoreductase
(complex II), ubiquinone-cytochrome c oxidoreductase (complex III), cytochrome c oxidase
(complex IV), and ATP synthase (complex V). In addition, the respiratory chain requires 2 small
electron carriers, ubiquinone and cytochrome c.
Energy generation via ATP synthesis involves two coordinated processes: 1) electrons
(hydrogen ions derived from NADH and reduced flavin adenine dinucleotide) are transported along
the complexes to molecular oxygen, resulting in the production of water; and 2) simultaneously,
protons (hydrogen ions) are pumped across the mitochondrial inner membrane (from the matrix to
the intermembrane space) by complexes I, III, and IV. ATP is generated by the influx of these protons
back into the mitochondrial matrix through complex V (ATP synthase (27)).
Figure 2: Mitochondria and Mitochondrial respiratory chain.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7447
Karu et al. (2004) proposed that biological effects of visible and near-IR light, in mammalian
cells, are initiated via the mitochondrial signaling pathway ⁽³⁵⁾. The authors also suggested that the
redox absorbance recorded in the spectral range close to 600900 nm changes in living cells (28).
These observations are of particular interest because a growing number of recent scientific
reports and observations are providing more support to the concept that the functions of
mitochondria go beyond the generation of ATP and the regulation of energy metabolism (29).
It has been shown that mitochondria are playing a major role as an integrator of intrinsic and
extrinsic cellular signals, which can affect the health and survival of the cell; as well as, may play an
essential role in the cell-to-cell communication signaling mechanisms (30). ATP binding to the
extracellular surface of P2Y-purinergic receptors, allows release of cytosolic Ca2+calcium, initiates
regulatory gene expression and induces a cascade of intracellular molecular signaling (31).
Both Ca2+ uptake and efflux from mitochondria consume the mitochondrial membrane
potential (ΔΨmt); and, in this way modify the mitochondrial activity (and therefore the ATP
synthesis), which can be regulated by LLLT.
From a clinician’s point of view (for selection of the most effective parameters and
appropriate LLLT’s medical devices) this scientific data provides a new practical guideline and
assistance towards a better understanding of the biomolecular mechanisms of LLLT. Recent reports
have demonstrated that ATP is a critical signaling molecule that allows cells and tissues throughout
the body to communicate with one another (32).
This new aspect of ATP as an intercellular signaling molecule allows broadening of the
understanding of the cellular and molecular mechanisms of LLLT, and hence provides grounds for
optimization of LLLT efficacy. It has been shown that neurons and other cells may release ATP into
muscle, gut, and bladder tissue as a messenger molecule. The specific receptors (a family of P2,
purinergic receptors) for ATP signaling were found and identified (31; 32).
ATP activation of P2 receptors (subtypes P2X and P2Y) can produce various biological
effects. A recent publication by Anders et al. (2008), demonstrated that P2Y2 and P2Y11 receptors
were expressed in near-IR light irradiated normal human neural progenitor cells in vitro (33). These
observations support the notion that the irradiation of cells with near-IR light in the interval close to
905 nm could be functioning as a surrogate for growth factors. Recent reviews indicate that
laboratories worldwide are now racing to turn the data about ATP as a neurotransmitter into
therapies (31).
As a neurotransmitter, ATP is directly involved in brain function, sensory reception, and the
neuron system control of muscles and organs. When released by neuronal and non neuronal cells, it
often triggers protective responses, such as bone building and cell proliferation (17; 39). The LLLT-
induced ATP’s signaling mechanism also is believed to be involved in its use during pain therapy
(29); it is needless to say that chronic and neuropathic pains are the first disorders treated
successfully with LLLT many decades ago.
The role of ATP as a signaling molecule provides a new basis for explaining the versatility of
LLLT effects (34). Numerous reports indicate that light could regulate gene expression via
mitochondrial mechanisms. For example, Hu et al., (2007) have shown that mitochondria play a
central role in cellular homeostasis, and their homeostatic center is the mitochondrial membrane
potential (mt). The assessment of the mt in cells conducted by the authors indicated that A2058 cells
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7448
treated with dosages higher than 0.5 J cm-2 He-Ne laser irradiation at 633 nm exhibited a marked
increase in mt. They also found upregulation of cytochrome c oxidase activity, increased
phosphorylation of Jun N-terminal kinase (JNK) and later, activated activator protein-1 (AP-1),
which led to increased cell proliferation (35).
Mitochondrial membrane potential may play a role in response of cells to light therapy.
Huang et al. (2004) used tetramethyl rhodamine methyl ester to compare the mitochondria of six
different cell types. They found that the mean mt differed significantly between cell types, but that
the cell area or size also differed, and that a more accurate comparison was to calculate the
integrated mt over the cell cross sectional area. Although fibroblasts had a high measured value of
mean mt, when integrated over the whole cell the value was actually the lowest of the cell types
tested because fibroblasts had a much greater surface area (36).
Neuronal cells had higher mean mt values in the cell bodies compared to the growth cones.
Further work is necessary to determine if mean mt or mean mt per cell area correlated with
measures of cellular response to light. However, there is more supportive evidence that cell types
with greater overall mitochondrial activity (for instance cortical neurons and cardiomyocytes) do in
fact respond well to light. The presented observations may suggest that cells with a high level of
mitochondrial activity (or high mt) may have a higher response to light than cells with low
mitochondrial activity (37).
The majority of authors agree that mitochondria are the key cellular targets, able to initiate
LLLT induced biologic events, leading to: upregulation of ATP production, induction of various
transcription factors and modulation of pro and anti-inflammatory gene expression. These effects in
turn may lead to cell biostimulation, modulation of the levels of various inflammatory mediators
(such as cytokines), release of the growth factors, as well as, increase in tissue oxygenation and
reparation. The results of these biological interactions in animals and humans include such
therapeutic effects as: acceleration in healing of chronic wounds, improvements in sport injuries,
reduction of inflammation and pain in arthritis and neuropathies, amelioration of toxicity and tissue
damage after infectious diseases and heart attacks, and restoration of vascular and neuronal damage
THE ROLE OF THE 905 nm NIR COHERENT (LASER) LIGHT IN LLLT: An alternative explanation
for LLLT effects (particularly in the near-IR wavelength region) on cells and mitochondria does not
rely on specific photon absorption by proteins and/or enzymes and their defined absorption bands
leading to photochemistry, but relies more on specific photon absorption leading to localized
photothermal effects, confined to sensitive structures, such as lipids through non-specific photon
absorption. The quantum energy of photons at 905 nm is only ~1. 3 eV and hence insufficient to
achieve an electronic excitation in most biomolecules, including cytochrome c oxidase, making a
vibrational or thermal activation of these target molecules more likely at this wavelength.
Since the total energy per photon delivered is small the resulting rise in average tissue
temperature would be insignificant if this energy was evenly distributed over the whole cell.
However in the case of coherent laser light the energy is not evenly distributed, but forms a speckle
pattern at any given instance in time. When a surface is illuminated by a light wave, according to
diffraction theory, each point on an illuminated surface acts as a source of secondary spherical
waves. Laser speckle is formed by interference (either constructive or destructive) of waves that
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7449
have been scattered from each point on the illuminated surface. If the surface is rough enough to
create path length differences exceeding one wavelength, giving rise to phase changes greater than
2_, the amplitude, and hence the intensity, of the resultant light varies randomly, from 0 to twice the
average intensity. As tissue is not static these speckle pattern are varying over time and after less
than 1/100th of a second the temporal average of the intensity is again established across the tissue.
If light of low coherence (i. e. made up of many wavelengths moving out of phase) is used, a speckle
pattern will not normally be observed, because the speckle patterns produced by individual
wavelengths have different dimensions and will normally cancel one another out. The "size" of the
speckles is a function of the wavelength of the light, the surface area of the laser beam that
illuminates the first surface, and the distance between this surface and the plane where the speckle
pattern is formed. In tissue, the diameter of the speckles is of the order of the wavelength of light i. e.
about 1 micron and thus comparable to the size of the mitochondria. While continuous wave light
sources speckle pattern are averaged temporally as stated above they do not do so if the light pulse
is shorter than the speckle relaxation time. Thus the hypothesis is that laser speckles produce by
super pulsed laser sources can produce micro-thermal gradients due to inhomogeneous energy
absorption that can stimulate or otherwise alter the metabolism within mitochondria (39).
This hypothesis would explain reports that some LLLT effects in cells and tissues are more
pronounced when coherent pulsed laser light is used than comparable non-coherent light from LED
or filtered lamp sources that is of similar wavelength range although not monochromatic (40). For
these micro-thermal gradients to be effective the pulse repetition rate needs to be slow enough to
allow a complete relaxation of these gradients (for micron sized ‘hot spots’ this relaxation time is in
the millisecond range) prior to the generation of a novel speckle pattern generated micro-thermal
Lilge, et al., (2009) used near-IR light at 905 nm (Theralase Inc.) and a high peak power of
50W cm-2, suggested a role of localized thermal activation (or selective photothermolysis) within
mitochondria in the mechanisms of super pulsed 905nm LLLT (5;48;49).
The authors suggest that selective photo thermolysis occurs if photon absorption within a
target structure is much higher than in the surrounding tissue, but the pulse duration is short
compared to the thermal relaxation time of the target structure. Two hundred nanosecond pulses of
905 nm laser light, used by the investigators, exceed the thermal relaxation time of structures
smaller than 200 nm. Hence, according to the authors, the cell membrane (20 nm) would radiate too
much energy to the surrounding area not the thermal energy and thus not achieving elevated
temperatures, hence presenting an unlikely target structure.
Huttmann and colleagues (1999) suggested that the inverse of the repetition rate of 1/10
kHz or 100 microseconds needs to be equivalent to at least several thermal relaxation times of the
target structure, permitting their cooling between pulses to avoid accumulation of heat, which would
finally exceed the Arrhenius damage integral (41). Therefore, the target structure would be required
to be smaller than 3 to 5 μm, but larger than 200 nm.
Mitochondria membranes have high amount of lipids with peak absorbance at 900 to 930nm,
close to the super pulsed 905 nm wavelength used. Their inherent size (a hydrophilic head group <,
1. 0 nm; and a hydrophobic core of the bilayer is typically 3. 0- 4. 0 nm thick,) and the possibility for
lipids to be the main absorber make mitochondria lipid structures the most apparent targets for
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7450
near-IR-induced selective photo thermolysis (6). Note these micro-thermal gradients and selective
photo thermolysis effects exist only for super pulsed light sources.
LLLT exhibits diverse effects on pathophysiologic processes according to varying
wavelengths and wave modes. It is could be suggested that the LLLT biomechanisms induced by 905
nm near infrared light, particularly upregulation by the nitric oxide pathway following the 905 nm
wave in the super pulsed mode, are not the same when compared to visible light.
Several additional scientific studies suggest that mitochondria are accountable for the
cellular response to LLLT. Passarella et al., (1984) reported that LLLT affect both transport and
enzymatic processes in cells by acting on the cell’s mitochondria and increasing the ATP synthesis
via oxidative phosphorylation (42).
Taken together, this clinical and scientific data strongly indicate the essential role of near- IR
laser light in the biomechanisms of LLLT healing. It can also be concluded that local transient heating
of particular cellular micro regions with a size characteristic of mitochondria based chromophores is
possible and this effect may contribute to the efficacy of LLLT. The local transient rise in
temperature may cause structural (conformational) changes of near-IR light absorbing molecules
and trigger secondary biochemical and other biomolecular activity such as activation or inhibition of
enzymes and gene expression. The opportunity of local transient heating of absorbing
chromophores, which is offered by the 905 nm super pulsed technology, takes medical applications
of modern LLLT to entirely new levels and clinical significance when whole anatomical tissues are
irradiated during patient treatment.
THE ROLE OF THE 660 nm COHERENT (LASER) RED LIGHT IN LLLT: In addition to the near-
infrared light at super pulsed 905 nm wavelengths, the most highly LLLT technologies encompasses
biomechanisms mediated by coherent visible red light at 660 nm wavelength or approximately 1. 9
eV per photon.
The most recent, well defined, in-vivo data suggests the presence of biological mechanisms
mediated by 660 nm laser light. Studying the effect of LLLT on inflammatory pain, the authors have
shown that Low-Level Laser phototherapy [InGaAIP visible laser diode (660 nm) with a radiant
exposure of 2. 5 J cm-2] was proven to be an effective analgesic, while non-coherent LED light did
not show a similar effect (22). Similar to the 905 nm speckle pattern exists a comparable time albeit
at smaller dimensions for 660 nm red laser light. Increased RNA and protein synthesis was
demonstrated after HeNe laser (632. 8 nm) treatment at 5 J cm-2 (43).
In 1994, Pastore et al (2004), found increased activity of cytochrome c oxidase and an
increase in polarographically measured oxygen uptake after 2 J cm-2 of HeNe (633 nm) irradiation.
As a major stimulation in the proton pumping activity, an increase of about 55% in the H+/e- ratio
was found in illuminated mitochondria indicating a role of the mitochondrial changes in the
mechanisms of red laser light based therapy (44).
W. Yu et al., 1997, used 660 nm laser at a power density of 10 mW cm-1 and showed
increased oxygen consumption following (0. 6 J cm-2 and 1. 2 J cm-2) radiance exposure, increased
phosphate potential, and energy charge (at 1. 8 J cm-2 and 2. 4 J cm-2) and enhanced activities of
NADH: ubiquinone oxidoreductase, ubiquinol: ferri cytochrome c oxidoreductase and
ferrocytochrome c : oxygen oxidoreductase (between 0. 6 J cm-2, and 4. 8 J cm-2) (45).
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7451
Originally, Gordon and Surrey (1960) found that cells exposure to light of 660 nm showed
enhanced ATP synthesis and may alter the phosphorylative capacity in mitochondria upon direct
exposure of the organelles in this red wavelength diapason. (46; 47).
In 2005, these original observations were confirmed by Amat et al., (48), and very recently
(2011) by Alghamdi and colleagues (49).
Yu et al., (1997) studied whether oxidative metabolism and electron chain enzymes in liver
mitochondria can be modulated by 660 nm wavelength photo irradiation. The oxygen consumption,
phosphate potential, and energy charge of liver mitochondria were determined following photo
irradiation (45).
It is generally believed that mitochondria are the primary affected organelle in mammalian
tissue irradiated with laser light at 660 nm and/or 905 nm(59; 60) albeit, possibly different
mechanisms exist ranging from direct photochemical to photothermal initiated effects.
This assumption led to the development of the highest efficacy advanced LLLT systems that,
as described in detail below, encompass potent and complementary bioregulatory mechanisms of
660 nm (red) laser light and 905 nm (near infrared) super pulsed laser light at a particular
therapeutic mode of administration.
There is certainly more than one reaction involved in the primary mechanisms of LLLT.
There are no grounds to believe that only one of these processes occurs when a tissue is irradiated.
An important question for the future research is which one of these reactions is responsible for a
certain wavelength effect. However, experimental data clearly support use of 660 nm visible red
laser light and 905 nm super pulsed NIR laser light based on their role in modulation of redox
mitochondrial function, changes in properties of terminal enzymes and cellular signaling that might
be critical steps in bioregulatory mechanisms of LLLT.
THE OPTICAL AND THERAPEUTIC WINDOWS OF LLLT: It should be noted that both absorption
coefficients (μa) and scattering (us) coefficients of light in tissue are wavelength dependent in any
given tissue. The light scattering is much more pronounced in the blue region of the visible spectrum
than in the red and near infrared spectrum (50).
The data suggests that, particularly in living tissues, red light penetrates deeper than blue
light, and the penetration of infrared light is deeper than red light. In addition, water in tissues
begins to absorb significantly at wavelengths greater than 960 nm, whereas wavelengths below
600nm are strongly absorbed by the hemoglobins (oxy HB, and deoxy HB) in the blood.
Therefore, although light at violet blue, green, yellow, and orange, wavelengths may have
various effects on cells growing in optically transparent culture medium, the use of LLLT in animals
and patients almost exclusively involves light in the range above 600 nm and below 950 nm with the
maximum effective “optical window” ranging from 650 nm to 930 nm (Figure 3).
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7452
Within the optical window of light transmission it is important to consider also the change in
quantum energy per photon that is dropping from ~ 1. 8 eV at 660 nm to ~ 1. 2 eV at 950 nm
respectively. While quantum energy above 1. 5 eV can result in electronic activation of biomolecules
and thus initiate direct photochemical events; this is not the case at lower quantum energies, and
activation energies below 1. 5 eV cannot result in direct photochemistry. Therefore, the change in
quantum energy per photon is a very important characteristic that may augment the LLLT clinical
efficacy (51; 52).
Hence, the medical device parameters are interrelated according where power densities
necessary for stimulation I stim have to surpass the threshold irradiance I (53; 54).
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7453
According to Sommer et al., (2001) (54) Light power densities lower than the threshold
values I obviously do not produce therapeutic effects, even under a prolongation of the irradiation
time. The effective range is given by the particular Arndt-Schultz curve similar to that suggested by
Sommer and colleagues (54). Although equation 3 is physically surprisingly simple, the biological
implications are by no means trivial. Biologically, the parameters and I stim are clearly independent
from each other, an important consideration at least for the medical applications of photobiological
effects with the use of lasers (including incoherent light sources) at low energy density levels (54).
BIPHASIC DOSE RESPONSE IN LOW LEVEL LIGHT THERAPY: One of the key factors that affect the
efficacy of LLLT is a biphasic dose response that directly correlates with the characteristics of the
wavelength and the amount of energy absorbed by a chromophore or a photoacceptor which of the
function of the light energy density (the radiant exposure or light fluence measured below the
The important role of a biphasic response has been demonstrated by a number of studies in
cell culture (55), animal models (21) and in clinical studies (56).
It has generally been observed that an optimal light irradiance and radiance exposure exists
for any particular application, and irradiances lower than this optimum value, or more significantly,
larger than the optimum value will have a diminished therapeutic outcome. Therefore for high doses
of light (a common disadvantage of Class IV Lasers used as a therapeutic means that are able to
produce an excessive amount of heat and /or generate an overwhelming level of power density
deposited into the tissue) a negative outcome may even result (56), known as bioinhibition.
In connection with the versatility of LLLT effects, and because of the probable existence of a
non-linear, biphasic power density response, as mentioned above, choosing the correct LLLT
medical device (in terms of irradiation parameters, such as: wavelength, irradiance, pulse structure,
coherence, polarization and dose characteristics) for any specific medical condition is mandatory to
achieve clinically efficacious results.
In addition, there has been some confusion in the literature about the delivered fluence when
the light spot is small. For example, according to Hamblin and colleagues, if 5 Joules of light is given
to a spot of 5 mm2 the fluence is 100 Jcm-2 which is nominally the same fluence as 100 Jcm-2
delivered to 10 cm2, but the total energy delivered in the latter case is 200 times greater, due to the
requisite increase in surface area.
Overall the concept of power density loses meaning rapidly when approaching small
irradiation spot sizes. In general, the light distribution in tissue is equal for any spot size diameter
smaller than 0. 5 mm, the inverse of the average reduced scattering coefficient μ’s reported for
tissues in the visible and near infra-red spectral range (57).
For light wavelengths greater than 650 nm, in most tissues and particularly in skin beam
surface area should be considered when the light spot approaches a diameter of ~ 2-3 mm as the
angular spread of the emitted light from the diodes could double this beam surface area prior to
reaching the tissue surface. In particular when laser diodes are clustered close together their beam
surface areas overlap combining the individual fluence rate profiles into a single profile within the
first few mm of depth into the tissue; hence, determining the effective area a can be more complex
than first imagined.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7454
Evidence suggests that both energy density (radiant exposure) and power density
(irradiance) are key biological parameters for the effectiveness of laser therapy, and they may both
operate within specific thresholds (i. e., : a lower and an upper threshold for both parameters
between which LLLT is effective, and outside of which LLLT is too weak [that is one of the common
therapeutic disadvantages for the light sources with power levels of less than 5 mW (i. e., many Class
I-II and IIIa light devices)] to have any effect or that intense energy densities is one of the common
therapeutic disadvantages for the light sources with energy density levels greater than 10 Jcm-2 (i.
e., : Class IV light devices) that the therapeutic effect is inhibited or can be in fact adverse to the
subject. (see figure 5) (54).
According to Hamblin and colleagues (2009), biphasic dose response can be represented by
the difference in integrated area under the curve of time tracking wound size in wounds treated with
LLLT compared to no treatment controls, with a clear maximum seen at 2 Jcm- 2, and a high dose of
50 Jcm-2 demonstrating a worsening of the wound healing time curve (36). In general, radiant
exposures of red or NIR light as low as 3 or 5 J cm-2 will be beneficial in-vivo, but a large dose such
as 50 or 100 Jcm-2 will lose the beneficial effect and most likely will become detrimental.
Therefore, the question is no longer whether light has clinical and safety efficacy, but rather
how energy from therapeutic low energy light devices (lasers or LEDs) is generated and what are the
most optimal light parameters for different applications of these light sources. LLLT, as all other
forms of therapeutic modalities regulated by the FDA has its active ingredients” or irradiation
parameters and therapeutic doses.
Figure 5: Biphasic dose response, based on data taken from (54)
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7455
THE IMPORTANCE OF PULSING IN LLLT (PULSE vs. CW): The efficacy of LLLT has been confirmed
by several hundred randomized, double-blinded, placebo-controlled clinical trials, and this
therapeutic method has been approved by the FDA, Health Canada and CE and numerous other
health regulatory agencies from dozens of countries worldwide (58).
Hence, the question is no longer whether LLLT has clinical effects but rather what are the
pertinent light parameters required to achieve the desired clinical effect for a particular clinical
indication. Both pulsed (PW) and continuous wave (CW) modes are available in LLLT devices, which
offer medical practitioners a wide range of therapeutic options, allowing them to exploit
photochemical effects versus photothermal or molecular biochemical effects.
One interesting observation is the favourable effect of 905 nm need to explain super pulsed
mode (SP) in pulse mode versus continuous wave mode (905 nm LLLT in SP mode is able to activate
iNOS transcription and translation, in an acute inflammation model, demonstrating the existence of a
different mechanisms of action and is dependent on the pulse duration, duty cycle and frequency of
the delivered photons.
In continuous wave applications, photons interact on an individual basis with the tissue
chromophores initiating a photochemical light-tissue interaction Super pulsed and pulsed sources
however must execute an additional non-photochemical interaction for biostimulation to take place.
A number of well designed clinical studies have reported similar results indicating a greater
efficacy of LLLT achieved with SP versus PW versus CW mode. For example sequentially pulsed
optical energy was reported to be more efficacious than CW mode in stimulating collagen production
in a suction blister model (59). Several other studies have also obtained a similar pattern of results.
In a wound healing study, Kymplova et al., (60) used a large number of women (n=2, 436) to
elucidate the effects of phototherapy on wound repair following surgical episiotomies (one of the
most common surgical procedures in women). A pulsed laser, used in the study, emitted light
(wavelength of 670 nm, power 20 mW and an energy density of 2 J cm-2) at various frequencies (10,
25, and 50 Hz) (the authors did not specified the pulse duration and duty cycle), and, according to
the authors, promoted wound repair and healing more effectively than a CW light source (60)
delivering a similar radiant exposure.
In a recent study by Brondon and colleagues (61), the photo-irradiation outcomes, following
delivery of 670 nm (10 mW cm-2, 5 J cm-2) light through a 0. 025% melanin filter via continuous
illumination or pulsed delivery at frequencies of 100 and 600 Hz, were examined. Pulsing had a
significantly greater stimulatory effect on cell proliferation and oxidative burst as compared to the
continuous photoradiation group (61).
Taken together, these findings underline the importance of pulsing parameters in LLLT and
how critical the implementation of optimal pulse duty cycle and peak power is to achieve maximal
stimulation of photo bioregulatory effects in targeted tissues.
Indeed, pulsing frequency (pulse repetition rate and pulse duration) appears to hold such
differential effects, as suggested by studies using different in vitro and in vivo models (62). These
observations have been confirmed by Barolet D, et al., (2010). The authors have shown that certain
pulsing frequencies appear to be more efficacious than others in triggering desired biological
outcomes (63).
The scientific evidence continues to demonstrate that pulsed light does have biological and
clinical effects that are different from those of continuous wave light. Several studies revealed that
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7456
LLLT in a PW mode of operation is substantially better able to penetrate through the melanin and
other skin barriers, supporting the hypotheses that pulsing is beneficial in reaching deep target
tissue and organs.
In 2009, Lapchak P. A and Taboada L. D. reported that transcranial near infrared laser
therapy (NILLLT) improves behavioural outcome following embolic strokes in embolized rabbits
and clinical rating scores in acute ischemic stroke (AIS) patients.
It was proposed that mitochondrial energy production may underlie this therapeutic
response to the near-infrared LLLT (NILLLT). The authors evaluated the effect of NILLLT on cortical
ATP content using the rabbit small clot embolic stroke model (RSCEM), the model originally used to
demonstrate NILLLT efficacy. In addition, the authors compared the effect of CW and PW
illumination on ATP production in the brain tissue.
Five minutes following embolization, rabbits were exposed to 2 min of NILLLT using an 808
nm laser source, which was driven to output 262. 5mW cm-2 either CW, or PW using 2 ms pulses at
100Hz. Three hours after embolization, the cerebral cortex was excised and processed for the
measurement of ATP content using a standard luciferin-luciferase assay.
NILLLT-treated rabbits were directly compared to sham-treated embolized rabbits and naive
control rabbits. Embolization decreased cortical ATP content in the ischemic cortex by 45%
compared to naive rabbits, a decrease that was attenuated by CW NILLLT which resulted in a 41%
increase in cortical ATP content. However, this net tissue content of ATP increase was not
statistically different from either the sham-treated embolized control group or the naive control
group (p= 0. 1357 and 0. 0826, respectively).
The results of the NILLLT in PW mode were much more compelling.
Following PW NILLLT, which delivered 35 times higher peak fluence rate than CW, the
authors reported a 221% (p=0. 0001) increase in cortical ATP content, compared to the sham
embolized group and naive control rabbits (p= 0. 0028).
The data undeniably demonstrates the benefit and the advantageous effect of pulsing in
LLLT and is providing a new insight into the molecular mechanisms associated with the LLLT clinical
efficacy (64).
In addition, as indicated above in association with equation 3, I stim the energy density, E/a
(Jcm⁻²) and the fluence rate (Wcm⁻²) are important parameters. While light delivery either as PW or
CW may not impact the energy density attainable at a given depth throughout the therapy session,
the power density can be increased depending on the laser duty cycle and thus I stim can become
larger than Iⁿ at greater depth.
The attainable depth of the LLLT bioregulation increase is based on the duty cycle of the
laser or the ratio of the light pulsed duration over dark period. Sinusoidal modulated light sources
typically have only a duty cycle of 0. 5 and hence an e⁻⁰⁵ depth advantage of only 2, whereas the duty
cycle of a 200 nsec pulse at 10Khz is 0. 005 and the depth advantage can be 200 to attain the same
fluence rate.
The overall body of literature on the subject has strongly indicated that clinical practitioners
must not underestimate the importance of energy rate and pulse structure when using different
LLLT sources. Therefore it is imperative that clinical and scientific researchers provide an
explanation of how does pulsed LLLT differ from the CW LLLT on the biomolecular level, how these
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7457
biomolecular changes are important for beneficial clinical outcomes of LLLT and what pulse
parameters are critical for photobioregulation.
They should also specifically determine what is the ideal pulse repetition rate or frequency
to be used, as the pulse rate may also have effects on the LLLT’s mediated biological responses.
THE CONCEPT OF SUPER PULSING: Both continuous wave (CW) and continuous wave light sources
that are turned on and off (pulsed light sources) cannot compare with the depth of penetration of
photonic energy delivered from super pulsed lasers.
Traditionally, cold lasers deliver photonic energy from diodes that range in power from
5mW to 500mW per diode; otherwise known as Class 3B laser devices. 200mW/cm2 is the highest
level of irradiance allowed in the visible for a continuous wave laser (Maximum Permissible
Exposure or MPE), because any greater power would result in excessive heat that could compromise
the safety and the efficacy of the Low Level Laser Therapy (LLLT) treatment. However, Super Pulsed
Laser can safely deliver optical energy from diodes that range in power from 10,000 mW to 50,000
mW peak power per diode.
This is paramount because the probability for a photon absorption event to occur largely
depends on the photon density or fluence rate and the photon absorption cross section of the
molecule where the former may be influenced by the pulse repetition rate and the peak power. In
superficial tissue, where the photon density is high, it is easy to reach the necessary power density
thresholds without the benefits of a super pulsed laser, in fact continuous wave lasers, pulsed lasers
or even LEDs can be utilized. However, in deep tissue where the photon density is extremely low, the
probability of photon absorption from individual pulsed laser speckles is extremely rare thus the
probability of reaching the necessary power density thresholds, I, is not possible for continuous
wave lasers, pulsed lasers or LEDs. However, super pulsed laser sources, through their intense
pulses of light can still overcome Iand photo chemically activate molecules at much deeper tissue
depths, This effect may also explain why head-to-head comparisons between lasers and LEDs in
deep tissue seem to be in favour of lasers and super pulsed lasers in particular (65).
Super pulsing allows for deeper penetration than an equivalent light source of the same
wavelength that is not super pulsed but has the same average output power. This is primarily
because short pulses can lead to a temporally higher concentration of excited chromophores at
greater depth which in turn can impact the biochemistry at the cellular level; whereas, the period
between pulses promotes optimization of thermal relaxation time of the target structure. This
relaxation time, permits cooling between pulses to avoid accumulation of heat and to avoid
detrimental effects, as well as to enhance the photochemical and photo physical light tissue
reactions, leading to superior observant therapeutic outcomes, such as: a reduction in inflammation,
elimination of pain and accelerated tissue healing.
Theralase’s super pulsed infrared laser light (905 nm) is Health Canada, FDA and CE Mark
certified and is in use in over 20 countries around the world. The super pulsed laser has the
distinction of being one of the fastest in the world - delivering pulses at 200 billionths of a second,
producing average powers of 100mW and peak powers up to 50, 000 mW per diode. The result is a
higher concentration of light power, or photons density, exceeding I deeper into the target tissue,
without the risk of burning the tissue.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7458
While CW and standard pulsed lasers are limited to less than 1-2 cm tissue depth for
therapeutic efficient penetration, super pulsed infrared laser technology is able to show therapeutic
efficacy at up to 10 cm into tissue and affect deeper target structures such as: bones, tendons,
ligaments and cartilage.
In our clinical and scientific experience, 905 nm super pulsed technology has been proven to
be more effective than 905 nm laser light treatment administered in a continuous wave mode (66).
TYPES OF PULSED LIGHT SOURCES: Five major types of pulsed lasers (or other light sources) are
commonly utilized: 1) Q switched, 2) Gain-switched, 3) Mode-locked, 4) Super pulsed and 5)
Chopped or gated. Each utilizes a different mechanism to generate light in a pulsed as opposed to
continuous manner, and vary in terms of pulse repetition rates, energies, and durations. However
the first three classes of pulsed lasers mentioned above are in general not used for LLLT; instead
super pulsed (a ‘‘true’’ pulsed laser) or gated lasers are mainly used. Moreover, the chopped or gated
light sources cannot compress available power into very short pulses, and hence are inefficient in
converting electrical power into LLLT’s optical power for very higher power densities.
SUPERPULSING OR A “TRUE” PULSED LLLT APPROACH: The concept of super pulsing was
originally developed for the carbon dioxide laser used in high power tissue ablative procedures.
The idea was conceived that by generating relatively short pulses (few milliseconds) within
the laser media more atoms can be excited than normally allowed in CW mode where heat
dissipation constraints limit the maximum amounts of energy that can be stored in the lasing media.
With the original carbon dioxide super pulsed lasers, the short pulses would confine the thermal
energy in the tissue (by making the pulse duration less than the thermal diffusion time) reducing
collateral thermal damage to normal tissue (67).
The type of laser that particularly benefited from super pulsing is the Gallium-Aluminum-
Arsenide (GaAlAs) and the Aluminum-Gallium-Indium-Phosphide (AlGaInP) diode laser. This GaAlAs
laser has a wavelength in the region of 904 to nm and pulse duration as short as 100 to 200
nanoseconds can be achieved.
Another semiconductor laser amenable to super pulsing is the Aluminum-Gallium-Indium-
Phosphide (AlGaInP) diode laser. It emits light at a similar wavelength (905nm) as the GaAlAs diode
laser, producing very brief pulses (100 to 200 nanoseconds) of high frequencies (in the range of
kilohertz). These pulses are of very high peak powers (150 W) and an average power of 150mW to
650 mW.
Theoretically, super pulsed GaAlAs and AlGaInP lasers allow I to be exceeded at greater
depth without the unwelcome effects of CW (such as thermal injury), as well as allowing for shorter
treatment times, and, hence, offer advantageous dose delivery and clinical benefits without the
undesirable adverse effects, such as including tissue damage or unfavourable biphasic effects.
Super pulsed lasers produce very powerful, pulsed peak power in the Watt range, but the
duration of the peak is typically only 100 to 200 nanoseconds. A GaAlAs laser presenting a peak
power of 10 W typically has an average output of 20 mW due to its very low duty cycle.
The other major class of pulsed light sources used in LLLT are not true pulsed lasers, but
simply CW lasers (usually diode lasers) that have a pulsed power supply generated by a laser driver
containing an often sinusoidal pulse generator. This technology is described as ‘‘chopped’’ or ‘‘gated’’
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7459
or “pseudo”. It is also equally feasible to use pulse generator technology to pulse LEDs or LED arrays
(68) albeit the ability to produce 100s of nsec pulses has not been demonstrated.
light offers numerous potential benefits. Because there are ‘‘quench periods’’ (pulse OFF times)
following the pulse ON times true, pulsed lasers or super pulsed lasers generate less tissue heating.
In instances where it is desirable to deliver light to deeper tissues increased powers are needed to
provide adequate power density at the target tissue, so I stim > I o. This increased power can cause a
transient tissue heating, particularly at specific biological targets (as discussed above in the
mitochondrial mechanisms of LLLT) and in this instance pulsed light could be very important.
Whereas CW or PW-generated by not “true” pulsed lasers (including ‘‘chopped’’ or ‘‘gated’’ or
“pseudo” technology), particularly emitted by non-coherent (non laser light sources), causes an
undesirable increase in temperature of the intervening and target tissues or organ.
Moreover, a “true” pulsed light that is used in LLLT has been shown to cause no measurable
change in the temperature of the irradiated area for the same delivered energy density as other non-
pulsed therapeutic light sources (58).
In 2006, Ilic S., et al., found that pulsed light (peak power densities of 750mWcm-2)
administered for 120 seconds produced no neurological or tissue damage, whereas an equal power
density delivered by CW (for the same number of seconds) caused marked neurological deficits (69).
Aside from safety advantages true, pulsed or super pulsed light might simply be more
effective than CW. The ‘‘quench period’’ (pulse OFF times) reduces tissue heating; thereby, allowing
the use of potentially much higher peak power densities than those that could be safely used in CW.
The higher power densities can change the reaction balances in favour of a positive signaling effect
or through the creation of thermal micro gradients changing the analyte exchange across cellular
and subcellular membranes. For example, when CW power densities at the skin use equal or higher
than 2 Wcm-2, doubling the CW power density would only marginally increase the treatment depth
while potentially significantly increasing the risk of thermal damage; in contrast, peak powers of
equal or higher than 5 Wcm-2 pulsed using appropriate ON and OFF times might produce little, or no
tissue heating. The higher pulsed peak powers can avoid tissue heating problems and improve the
fluence rate at deep tissues, achieving greater treatment depths and hence better, overall
therapeutic efficacy (40; 63).
There may be other biological reasons for the improved efficacy of pulsed light (PW) over
CW. According to Hashmi J. T., et al., the majority of the pulsed light sources used for LLLT have
frequencies in the 2. 510, 000 Hz range and pulse durations are commonly in the range of a few
milliseconds. This observation suggests that if there is a biological explanation of the improved
effects of pulsed light it is either due to some fundamental frequency that exists in biological systems
in the range of tens to hundreds of Hz, or alternatively due to some biological process that has a time
scale of a few milliseconds (58). Indeed, the possibility of resonance occurring between the
frequency of the light pulses and the neuronal electromagnetic frequency may in some way explain a
number of the beneficial results with LLLT using true pulsed light (70).
In addition, there are several lines of evidence that ion channels are involved in the
subcellular effects of LLLT, playing the critical role in the intercellular signaling induced in tissues by
LLLT (71).
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7460
Some channels permit the passage of ions based solely on whether their charge is of positive
(cationic) or negative (anionic) while others are selective for specific species of ion, such as sodium
or potassium. These ions move through the channel pore single file nearly as quickly as the ions
move through free fluid. In some ion channels, passage through the pore is governed by a ‘‘gate, ’’
which may be opened or closed by chemical or electrical signals, temperature, or mechanical force,
depending on the variety of channel. Ion channels are especially prominent components of the
nervous system.
Voltage activated ion channels underlie the nerve impulse while transmitted, activated or
ligand gated channels mediate conduction across the synapses. There is a large body of literature on
the kinetics of various classes of ion channels but in broad summary it can be claimed that the time
scale or kinetics for opening and closing of ion channels is of the order of a few milliseconds.
For instance, there are several reports indicating that the diverse types of ion channel
receptors have kinetics with timescales that are comparable with the super pulsing technology (72;
73), as well potassium and calcium ion channels in the mitochondria and the sarcolemma may be
involved in the cellular response to LLLT (74; 75).
Several authors have also suggested that there is the possibility that one cellular mechanism
of action of LLLT is the photo dissociation of nitric oxide from a protein binding site (heme or copper
center) such as those found in cytochrome c oxidase. If this process occurs it is likely that the NO
would rebind to the same site even in the presence of continuous light. Therefore if the light was
pulsed multiple photo dissociation events could occur, while in CW mode the number of
dissociations may be much smaller (58).
THE LIGHT PENETRATION DEPTH: The most important parameter that governs the depth of
penetration of laser light into tissue is wavelength. When light at a specific wavelength interacts with
particular tissue, it is either absorbed or scattered in various proportions depending on the optical
properties of the tissue. The tissue characteristics are important for LLLT and all other forms of
phototherapy and medical laser applications, in order to understand the interaction mechanisms
between light and tissue. Knowledge concerning light transport in tissue and hence the composition
of tissue is essential for proper light dosimetry. In general, both the absorption (μa)and scattering
(μs)coefficients of living tissues are higher at lower wavelengths, so near-infrared light (up to about
950 nm) penetrates more deeply that visible and UV light. It is often claimed that pulsed lasers
penetrate more deeply into tissue than CW lasers with the same average power density (76).
Many applications of LLLT may require deep penetration of the light energy, such as deep
treated conditions like: lower back, neck, and hip joint pain and inflammation. Therefore, if the
power densities need to be greater than a few mWcm-2 and are required to be delivered safely to
target tissues > 5 cm below the skin surface, the effective treatment can only be achieved by using
super pulsed lasers (58).
THE USE OF COMBINED LASERS (CW + PW) IN LLLT: Scientific and clinical data suggest that a
relatively high fluence (the light energy density delivered to the target tissue) is necessary to
attenuate pain, whereas a lower fluence decreases inflammation. If this is indeed the case, for a
number of clinical conditions, including musculoskeletal applications, achieving higher doses at the
level of the target tissue may not be ideal.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7461
It has also been postulated that successful LLLT treatments of arthritic joints bring benefit
not by reaching the deep target tissue directly but by inhibiting superficial nociceptors. In other
words, the therapy brings relief primarily by attenuating pain perception, as opposed to reduction of
deep tissue inflammation (58).
It becomes evident that the use of a prescribed dose at the tissue surface is insufficient to
reach a desired target dose at depth, as the tissue thickness to the target differs between patients, as
do the tissue attenuation coefficients which are based on tissue compositions. Hence, new
technology is being developed to, optimize these variables and develop a smart) technology that
enables the determination of the power density, Istim and the fluence at the target tissue’s depth.
These scientific reports, as well as, observations on the complementary biological actions of
the far-red and near-infrared irradiation have led to the beginning of the research and development
of a unique class of platform technologies that combine (CW + PW and SP) LLLT approaches; and
after FDA and other health regulatory agencies approvals in Canada, Europe, Latin America and in
Asia, to a successful commercialization of the new and effective phototherapeutic medical devices.
The latest medical device technology in the field employs visible red (CW) and near infrared
super pulse (SP) proprietary technology to comprise a unique and clinically validated array of
multiple probe lasers. As a result of this synchronizing therapeutic approach, benefits offered by this
new technology includes: 1) delivery of a well-defined, particular dose of coherent photon energy
per treatment given to a patient and 2) implementation of cooperative stimulation of the proximal
and distal therapeutic mechanisms, in tissues, to induce bioregulatory responses that mitigate
inflammation and pain, as well as, the ability to accelerate the healing in the disease in affected areas
and assist in maintaining remission, particularly through ₍₃₎delivering energy at Istim > Io to the
required depth. Up until now, in the peer-reviewed literature, there has been remarkably little
information available about any laser technology that encompasses these concepts.
In Gigo-Benato’s study, CW or PW stand alone was compared to combined laser. In short, the
combined laser was found to be more effective in stimulating nerve regeneration, than either CW or
PW alone (77).
The two other studies used a combined laser (CW and PW) to administer laser acupuncture,
along with Transcutaneous Electrical Nerve Stimulation (TENS), to patients with symptoms of pain.
Naeser et al., administered this ‘‘triple therapy’’ to patients suffering from carpal tunnel syndrome
(CTS) (78).
Eleven patients with mild-to-moderate symptoms of CTS were selected to participate in the
trial. All participants were previously treated by various methods, but had failed to respond to
standard medical or surgical treatment regimens. Subjects were divided into two groups, one of
which received sham irradiation and the other received a combined treatment of LLLT (CW and PW)
and TENS. As compared to controls, the treated group experienced statistically significant
improvement and remained stable for 1to 3 years.
The results of this study indicate the therapeutic advances of combined LLLT in the
treatment of CTS. Ceccherelli et al., (1989) administered laser acupuncture to patients suffering from
myofascial pain (79). In this double blinded placebo controlled trial, patients received either the
same ‘‘triple therapy’’ as in the Naeser et al. study (CW, PW, and TENS) or sham irradiation, every
other day over the course of 24 days. Results were encouraging, with the treatment group
experiencing a significant improvement in symptoms, both immediately after the treatment regimen
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7462
and at a 3-month follow up visit. In both preceding studies, the combined regimen of CW, PW, and
TENS was compared to untreated controls, and found to be effective (78). However, neither study
compared CW and PW or administered CW, PW, or TENS individually. Therefore, it is impossible to
determine whether stand alone the combination of CW and PW light would have produced similar
results, or if the used combined LLLT regimen along with TENS was more effective.
As described above, the therapeutic optical windows (660 nm and 905 nm) utilized by The
latest LLLT technologies are correspond to the absorption and the action spectra optical windows of
the key mitochondria chromophores, such as cytochrome c oxidase and cellular membrane lipids.
Moreover, it is apparent that 660 nm and 905 nm light have an impact on the mitochondrial
chromophores via independent mechanisms and hence the combination of 660 nm and 905 nm light
is considered highly probable to have an additive biologic effect compared to individual
wavelengths. This biologic effect is amplified to achieve the effective therapeutic outcomes via
implementation of the latest LLLT technology cooperative CW and PW stimulation system that
target the proximal and distal therapeutic mechanisms, in tissues, to induce bioregulatory responses
that effectively modulate local and systemic pathologic manifestations in the LLLT treated patients.
Latest LLLT technology provides significant clinical symptoms relief and a quality of life
improvement in patients with osteoarthritis and untreated injuries to muscles, ligaments and
tendons that were enrolled into the study based on the Philadelphia Panel Classification, 2001 (80).
Considering the multifactorial etiopathogenesis of the majority diseases, it is likely that the
efficacy of LLLT is determent by optimal sets of therapeutic wavelengths, which should be optimized
based on the optical properties of treated tissues and regiments of light delivery targeting particular
chromophores at specific tissue depth.
1. Comparison of four non-invasive rewarming methods for mild hypothermia. Daanen, H. A.
and Van de Linde, FJ. 12, s. l. Aviat Space Environ Med, 1992, Vol. 63, pp. 1070-1076.
2. Transcutaneous electrostimulation for osteoarthritis of the knee. Rutjes, AW, et al. 4, Oct
2009, Cochrane Database Syst Rev, Vol. 7.
3. Tennis elbow. Clinical Evidence. Bisset, L, Coombes, B and Vicenzino, B. [Online] june 27,
2011. [Cited: June 27, 2009.] http://clinicalevidence. bmj. com/ceweb/index. jsp.
4. Safety of Lase Products user's guide. s. l. Commission, International Electro technical. IEC
International Electro technical Commission, 2004. TR 60825-14.
5. Low power laser treatment in patients with knee osteoarthritis. Tascioglu, F, et al. 17-18,
May 1, 2004, Swiss Med Wkly, Vol. 134, pp. 254-258.
6. In vivo effects of low level laser therapy on inducible nitric oxide synthase. Moriyama Y,
Nguyen J, Akens M, Moriyama EH, Lilge L. 3, March 2009, Lasers Surg Med, Vol. 41, pp. 227-
7. Photo dissociation of cytochrome c oxidase-nitric oxide complexes. Gorren AC, Van Gelder
BF, Wever R. 1988, Ann N Y Acad Sci, Vol. 550, pp. 139-149.
8. Mitochondrial nitric oxide synthase: current concepts and controversies. Lacza Z, Pankotai E,
Busija DW. 14, Jan 2009, Front Biosci, Vol. 1, pp. 4436-4443.
9. Nitric oxide and mitochondria. GC. Brown. 12, Jan 2007, Front Biosci, Vol. 1, pp. 1024-1033.
Journal of Evolution of Medical and Dental Sciences/ Volume 2/ Issue 39/ September 30, 2013 Page 7463
10. Redox signaling (cross-talk) from and to mitochondria involves mitochondrial pores and
reactive oxygen species. A, Daiber. 6-7, Jul 2010, Biochim Biophys Acta, Vol. 1797, pp. 897-
1. Samith Ahmed
2. Gregory Bewsh
3. Shankaranarayana Bhat
4. Ramesh Babu
1. Consultant Physician, Department of
Alternative Medicine, Sree Sanjeevini
2. Research Manager, Department of Alternative
Medicine, Sree Sanjeevini Solutions.
3. Branch Manager, Department of Alternative
Medicine, Sree Sanjeevini Solutions.
4. CEO, Department of Alternative Medicine, Sree
Sanjeevini Solutions.
Dr. Samith Ahmed,
Sree sanjeevini cold laser cafe-
No 316, 3rd floor Sree Krupa 15th cross opp to
Karur vysya bank
Sampige road, Malleswarm
Date of Submission: 03/09/2013.
Date of Peer Review: 07/09/2013.
Date of Acceptance: 20/09/2013.
Date of Publishing: 24/09/2013
... It also promotes angiogenesis and synthesis of collagen [21][22][23][24]. Specifically, the use of low-level laser therapy (LLLT) for wound healing, nerve injury repair, as well as the reduction of inflammation and pain, was tested shortly after the invention of lasers [25]. Photobiomodulation using laser technology is often referred to as LLLT because the energy delivered to the tissue is much lower than for other laser treatments and it does not produce heating of tissue [25]. ...
... Specifically, the use of low-level laser therapy (LLLT) for wound healing, nerve injury repair, as well as the reduction of inflammation and pain, was tested shortly after the invention of lasers [25]. Photobiomodulation using laser technology is often referred to as LLLT because the energy delivered to the tissue is much lower than for other laser treatments and it does not produce heating of tissue [25]. In this context, Pinheiro et al. [23] demonstrated that bone irradiated with low-level lasers showed increased osteoblastic and collagen proliferation, as well as new bone formation in comparison with nonirradiated groups. ...
Full-text available
Background: Alveolar bone defects are usually the main concern when planning implant treatments for the appropriate oral rehabilitation of patients. To improve local conditions and achieve implant treatments, there are several methods used for increasing bone volume, among which one of the most successful, versatile, and effective is considered to be guided bone regeneration. The aim of this demonstrative study was to propose an innovative analysis protocol for the evaluation of the effect of photobiomodulation on the bone regeneration process, using rat calvarial defects of 5 mm in diameter, filled with xenograft, covered with collagen membrane, and then exposed to laser radiation. Methods: The animals were sacrificed at different points in time (i.e., after 14, 21, and 30 days). Samples of identical dimensions were harvested in order to compare the results obtained after different periods of healing. The analysis was performed by cross-linking the information obtained using histology and high-resolution synchrotron-based tomography on the same samples. A comparison was made with both the negative control (NC) group (with a bone defect which was left for spontaneous healing), and the positive control (PC) group (in which the bone defects were filled with xenografts and collagen membrane without receiving laser treatment). Results: We demonstrated that using photobiomodulation provides a better healing effect than when receiving only the support of the biomaterial. This effect has been evident for short times treatments, i.e., during the first 14 days after surgery. Conclusion: The proposed analysis protocol was effective in detecting the presence of higher quantities of bone volumes under remodeling after photobiomodulation with respect to the exclusive bone regeneration guided by the xenograft.
Full-text available
Photobiomodulation therapy (PBMT) previously known as low-level laser therapy (LLLT) has been used for over 30 years, to treat neurological diseases. Low-powered lasers are commonly used for clinical applications, although recently LEDs have become popular. Due to the growing application of this type of laser in brain and neural-related diseases, this review focuses on the mechanisms of laser action. The most important points to consider include the photon absorption by intracellular structures; the effect on the oxidative state of cells; and the effect on the expression of proteins involved in oxidative stress, inflammation, pain, and neuronal growth.
Full-text available
The aim of this study was to investigate the analgesic efficacy of low power laser therapy in patients with knee osteoarthritis (OA). The study design was randomised, placebo-controlled and single blinded. Sixty patients with knee OA according to the American College of Rheumatology criteria were included and randomly assigned to three treatment groups: active laser with dosage of 3J/per painful point, active laser with a dosage of 1.5/J per painful point and placebo laser treatment groups. A Gal-Al-As diode laser device was used as a source of low power laser with a power output of 50 mW and a wavelength of 830 nm. The patients were treated 5 times weekly with 10 treatments in all. The clinical assessments included Western Ontario and McMaster Universities osteoarthritis index (WOMAC) pain, stiffness and physical function subscales. In addition, the intensity of pain at rest and on activation was evaluated on a visual analogue scale. Compared to baseline, at week 3 and at month 6, no significant improvement was observed within the groups. Similarly, no significant differences were found among the treatment groups at any time. With the chosen laser type and dose regimen the results that we obtained in this study, suggest that low-level laser therapy has no effect on pain in patients with knee OA.
Full-text available
Lateral pain in the elbow affects up to 3% of the population, and is considered an overload injury of the extensor tendons of the forearm where they attach at the lateral epicondyle. Although usually self-limiting, symptoms may persist for over 1 year in up to 20% of people. METHODS AND OUTCOMES: We conducted a systematic review and aimed to answer the following clinical question: What are the effects of treatments for tennis elbow? We searched: Medline, Embase, The Cochrane Library, and other important databases up to November 2009 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). We found 80 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. In this systematic review we present information relating to the effectiveness and safety of the following interventions: acupuncture, autologous whole blood injections, corticosteroid injections, combination physical therapies, exercise, extracorporeal shock wave therapy, iontophoresis, low-level laser therapy, manipulation, non-steroidal anti-inflammatory drugs (oral and topical), orthoses (bracing), platelet-rich plasma injections, pulsed electromagnetic field treatment, surgery, and ultrasound.
This review highlights the important role of redox signaling between mitochondria and NADPH oxidases. Besides the definition and general importance of redox signaling, the cross-talk between mitochondrial and Nox-derived reactive oxygen species (ROS) is discussed on the basis of 4 different examples. In the first model, angiotensin-II is discussed as a trigger for NADPH oxidase activation with subsequent ROS-dependent opening of mitochondrial ATP-sensitive potassium channels leading to depolarization of mitochondrial membrane potential followed by mitochondrial ROS formation and respiratory dysfunction. This concept was supported by observations that ethidium bromide-induced mitochondrial damage suppressed angiotensin-II-dependent increase in Nox1 and oxidative stress. In another example hypoxia was used as a stimulator of mitochondrial ROS formation and by using pharmacological and genetic inhibitors, a role of mitochondrial ROS for the induction of NADPH oxidase via PKCvarepsilon was demonstrated. The third model was based on cell death by serum withdrawal that promotes the production of ROS in human 293T cells by stimulating both the mitochondria and Nox1. By superior molecular biological methods the authors showed that mitochondria were responsible for the fast onset of ROS formation followed by a slower but long-lasting oxidative stress condition based on the activation of an NADPH oxidase (Nox1) in response to the fast mitochondrial ROS formation. Finally, a cross-talk between mitochondria and NADPH oxidases (Nox2) was shown in nitroglycerin-induced tolerance involving the mitochondrial permeability transition pore and ATP-sensitive potassium channels. The use of these redox signaling pathways as pharmacological targets is briefly discussed.
Osteoarthritis is the most common form of joint disease and the leading cause of pain and physical disability in the elderly. Transcutaneous electrical nerve stimulation (TENS), interferential current stimulation and pulsed electrostimulation are used widely to control both acute and chronic pain arising from several conditions, but some policy makers regard efficacy evidence as insufficient. To compare transcutaneous electrostimulation with sham or no specific intervention in terms of effects on pain and withdrawals due to adverse events in patients with knee osteoarthritis. We updated the search in CENTRAL, MEDLINE, EMBASE, CINAHL and PEDro up to 5 August 2008, checked conference proceedings and reference lists, and contacted authors. Randomised or quasi-randomised controlled trials that compared transcutaneously applied electrostimulation with a sham intervention or no intervention in patients with osteoarthritis of the knee. We extracted data using standardised forms and contacted investigators to obtain missing outcome information. Main outcomes were pain and withdrawals or dropouts due to adverse events. We calculated standardised mean differences (SMDs) for pain and relative risks for safety outcomes and used inverse-variance random-effects meta-analysis. The analysis of pain was based on predicted estimates from meta-regression using the standard error as explanatory variable. In this update we identified 14 additional trials resulting in the inclusion of 18 small trials in 813 patients. Eleven trials used TENS, four interferential current stimulation, one both TENS and interferential current stimulation, and two pulsed electrostimulation. The methodological quality and the quality of reporting was poor and a high degree of heterogeneity among the trials (I(2) = 80%) was revealed. The funnel plot for pain was asymmetrical (P < 0.001). The predicted SMD of pain intensity in trials as large as the largest trial was -0.07 (95% CI -0.46 to 0.32), corresponding to a difference in pain scores between electrostimulation and control of 0.2 cm on a 10 cm visual analogue scale. There was little evidence that SMDs differed on the type of electrostimulation (P = 0.94). The relative risk of being withdrawn or dropping out due to adverse events was 0.97 (95% CI 0.2 to 6.0). In this update, we could not confirm that transcutaneous electrostimulation is effective for pain relief. The current systematic review is inconclusive, hampered by the inclusion of only small trials of questionable quality. Appropriately designed trials of adequate power are warranted.
Low level laser therapy (LLLT) has been demonstrated to modulate inflammatory processes with evidence suggesting that treatment protocol, such as wavelength, total energy, and number of treatments determine the clinical efficacy. In this study, the effects of LLLT mediated by different wavelengths and continuous versus pulsed delivery mode were quantified in a transgenic murine model with the luciferase gene under control of the inducible nitric oxide synthase (iNOS) expression. LLLT modulated iNOS gene expressed in the acute Zymosan-induced inflammation model is quantified using transgenic mice (FVB/N-Tg(iNOS-luc)). Here an energy density of 5 J cm(-2) at either 635, 660, 690, and 905 nm in continuous wave mode and at 905 nm for short pulse delivery were evaluated. Age of the animals was determined as additional modulating the inflammatory response and the LLLT efficacy for some treatment protocols. Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression for some LLLT protocols. Intensity and time course of inducible nitric oxide expression was found to not only depend on wavelength, but also on the mode of delivery, continuous, or pulsed irradiation. LLLT exhibit different effects in induced inflammatory process according to different wavelengths and wave mode. Upregulation of iNOS gene following 905 nm pulsed wave suggests a different mechanism in activating the inflammatory pathway response when compared to the continuous wave.
New discoveries in the last decade significantly altered our view on mitochondria. They are no longer viewed as energy-making slaves but rather individual cells-within-the-cell. In particular, it has been suggested that many important cellular mechanisms involving specific enzymes and ion channels, such as nitric oxide synthase (NOS), ATP-dependent K+ (KATP) channels, and poly-(APD-ribose) polymerase (PARP), have a distinct, mitochondrial variant. Unfortunately, exploring these parallel systems in mitochondria have technical limitations and inappropriate methods often led to inconsistent results. For example, the intriguing possibility that mitochondria are significant sources of nitric oxide (NO) via a unique mitochondrial NOS variant has attracted intense interest among research groups because of the potential for NO to affect functioning of the electron transport chain. Nonetheless, conclusive evidence concerning the existence of mitochondrial NO synthesis is yet to be presented. This review summarizes the experimental evidence gathered over the last decade in this field and highlights new areas of research that reveal surprising dimensions of NO production and metabolism by mitochondria.
The dissociation of cytochrome c oxidase-nitric oxide complexes was studied by optical spectroscopy at cryogenic temperatures (15 degrees K). With the reduced cytochrome c oxidase-nitric oxide complex, the observations that were reported by Yoshida et al. were confirmed. Photodissociation of the oxidized cytochrome c oxidase-nitric oxide complex did not induce any significant absorbance changes between 350 and 875 nm. With the azide-nitrosyl-cytochrome c oxidase complex, the illumination caused the dissociation of the a2+(3).NO complex to the unligated state a2+(3). Increasing the temperature to 77 degrees K led to the formation of a new complex, probably a3+(3).N3-. The N3(-)-NO-cytochrome c oxidase complex was the only compound for which appreciable photodissociation was achieved by continuous illumination at room temperature (20 degrees C). The effect of illumination was biphasic. In the first phase the a2+(3).NO complex is dissociated and cytochrome a3 oxidized by an electron transfer to CuB. In the second phase nitric oxide, which is still bound to CuB after the first phase, is expelled from the complex by azide, with a concomitant electron transfer from CuB to cytochrome a.
Nitric oxide (NO) and its derivatives (reactive nitrogen species) have multiple effects on mitochondria that impact on cell physiology and cell death. Mitochondria may produce and consume NO and NO stimulates mitochondrial biogenesis, apparently via cGMP upregulation of transcriptional factors. NO inhibits mitochondrial respiration via: (A) an acute and reversible inhibition of cytochrome oxidase by NO in competition with O2, and (B) irreversible inhibition of multiple sites by reactive nitrogen species. NO is a potent vasodilator (via cGMP), increasing O2 and respiratory substrate supply to mitochondria. NO stimulates reactive oxygen and nitrogen species production from mitochondria via respiratory inhibition, reaction with ubiquinol and reaction with O2 in the membrane. NO can induce apoptosis, mainly via oxidative stress. NO induces necrosis, mainly via energy depletion. Reactive nitrogen species activation of the mitochondrial permeability transition pore may cause apoptosis or necrosis. NO may protect against mitochondria-mediated cell death by multiple mechanisms.
  • Mitochondria Nitric Oxide
  • Gc
  • Brown
Nitric oxide and mitochondria. GC. Brown. 12, Jan 2007, Front Biosci, Vol. 1, pp. 1024-1033.
Comparison of four non-invasive rewarming methods for mild hypothermia. Daanen, H. A. and Van de Linde, FJ. 12, s. l
Comparison of four non-invasive rewarming methods for mild hypothermia. Daanen, H. A. and Van de Linde, FJ. 12, s. l. Aviat Space Environ Med, 1992, Vol. 63, pp. 1070-1076.