Presentations by Professor Tiina Karu
Laser Therapeutics,
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T.I.
KARU
CELLULAR
MECHANISMS of LOW-POWER LASER THERAPY
(PHOTOBIOMODULATION)
1. What is
photobiomodulation (low-power laser therapy?)
More
than 30 year ago the first publications about low-power laser therapy
or photobiomodulation (at that time called laser biostimulation)
appeared. Since then approximately 2000 studies have been published on
this topic (analysis of these publications can be found in [1]).
Medical treatment with coherent light sources (lasers) or noncoherent
light (Light Emitting Diodes, LED's) has passed through its childhood
and early maturity. Photobiomodulation is being used by
physiotherapists (to treat a wide variety of acute and chronic
muscosceletal aches and pains), dentists (to treat inflamed oral
tissues, and to heal diverse ulcerations), dermatologists (to treat
oedema, indolent ulcers, burns, dermatitis), rheumatologists (relief
of pain, treatment of chronic inflammations and autoimmune diseases),
and by other specialists (e.g., for treatment of middle and inner ear
diseases, nerve regeneration). Photobiomodulation is also used in
veterinary medicine (especially in racehorse training centers) and in
sports medicine and rehabilitation clinics (to reduce swelling and
hematoma, relief of pain and improvement of mobility and for treatment
of acute soft tissue injuries). Lasers and LED's are applied directly
to respective areas (e.g., wounds, sites of injuries) or to various
points on the body (acupuncture points, muscle trigger points). For
details of clinical applications and techniques used, the books [ 1-3]
are recommended.
2. What light
sources (lasers, LED's) can be used?
The
field of photobiomodulation is characterized by variety of
methodologies and use of various light sources (lasers, LED's) with
different parameters (wavelength, output power, continuous wave or
pulsed operation modes, pulse parameters). These parameters are
usually given in manufacturers manuals.
The
GaAlAs diodes are used both in diode lasers and LED's, the difference
is whether the device contains the resonator (as the laser does) or
not (LED). In latter years, longer wavelengths (-800-900 nm) and
higher output powers (to 100 mW) are preferred in therapeutic devices.
Should
a medical doctor use a laser or a diode? The answer is - it depends on
what one irradiates, in other words, how deep tissue layers must be
irradiated. By light interaction with a biotissue, coherent properties
of laser light are not manifested at the molecular level. The
absorption of low-intensity laser light by biological systems is of a
purely noncoherent (i.e., photobiological) nature. On the cellular
level, the biological responses are determined by absorption of light
with photoacceptor molecules (see the section 3 below). Coherent
properties of laser light are not important when cellular monolayers,
thin layers of cell suspension as well as thin layers of tissue
surface are irradiated (Fig. 1). In these cases, the coherent and
noncoherent light (i.e., both lasers and LED's) with the same
wavelength, intensity and dose provides the same biological response.
Some additional (therapeutical) effects from the coherent and
polarized radiation (lasers) can occur in deeper layers of bulk tissue
only and they are connected with random interference of light waves.
An interested reader is guided to the ref. [4] for more details. Here
we illustrate this situation by Fig. 1. Large volumes of tissue can be
irradiated by laser sources only because the length of longitudinal
coherence Lcoh is too small for noncoherent radiation sources [4].
3. Enhancement of
cellular metabolism via activation of respiratory chain: a universal
photobiological action mechanism
A
photobiological reaction involves the absorption of a specific
wavelength of light by the functioning photoacceptor molecule. The
photobiological nature of photobiomodulation means that some molecule
(photoacceptor) must first absorb the light used for the irradiation.
After promotion of electronically excited states, primary molecular
processes from these states can lead to a measurable biological effect
(via secondary biochemical reaction, or photosignal transduction
cascade, or cellular signaling) at the cellular level. The
question is, which molecule is the photoacceptor.
When
considering the cellular effects, this question can be answered by action
spectra. Any graph representing a photoresponse as a function of
wavelength, wave number, frequency, or photon energy, is called action
spectrum. Action spectra have a highest importance for identifying the
photoacceptor inasmuch as the action spectrum of a biological response
resembles the absorption spectrum of the photoacceptor molecule.
Existence of a structured action spectrum is strong evidence that the
phenomenon under study is a photobiological one (i.e., primary
photoacceptors and cellular signaling pathways exist). Fig. 2
represents some examples of action spectra for eukaryotic cells: two
of them (A, B) consider the processes occurring in cell nucleus, and
one spectrum (C) is for cell membrane. Fig. 2D shows the absorption
spectrum of the monolayer of the same cells.
The
spectra in Fig. 2 represent the red-to-near infrared (IR) region only,
i.e. the region that is most important for photobiomodulation. The
action spectra for full visibleto-near IR region can be found in [5].
In [5] one can find action spectra for various cellular responses for
other eukaryotic and prokaryotic cells as well.
Two
conclusions can be drawn from action spectra in Fig. 2. First, the
similarity of the action spectra for different cellular responses
suggests that the primary photoacceptor is the same for all these
responses. Second, the existence of the action spectra for biochemical
processes occurring in various cellular organelles (nucleus, Fig. 2A,
B and plasma membrane, Fig. 2C) assume the existence of cellular
signaling pathways inside of a cell between the photoacceptor and the
nucleus as well as between the photoacceptor and cell membrane. Action
spectra also indicate, which wavelengths are the best for irradiation:
maximal biological responses are occurring when irradiated at 620,
680, 760 and 820-830 nm (maxima of the spectra in Fig. 2). Skipping
over the story of identifying the photoacceptor (described in [5]) let
us conclude that photoacceptor for eukaryotic cells in red-to-near IR
region is believed to be the terminal enzyme of the respiratory chain cytochrome
c oxidase (located in cell mitochondrion). To be more exact, it is
a mixed valence (partially reduced) form of this enzyme, which has not
yet been identified. In the violet-to-blue spectral region,
flavoproteins (e.g., NADHdehydrogenase in the beginning of the
respiratory chain) are also among the photoacceptors as well terminal
oxidases.
An
important point has to be emphasized. When the excitable cells (e.g.,
neurons, cardiomyocites) are irradiated with monochromatic visible
light, the photoacceptors are also believed to be components of
respiratory chain. Some of the experimental evidence concerning
excitable cells is shortly summarized in Fig. 3. It is quite clear
from experimental data (reviewed in [4]) that irradiation can cause
physiological and morphological changes in nonpigmental excitable
cells via absorption in mitochondria. Later, similar irradiation
experiments were performed with neurons in connection with low-power
laser therapy. It was shown in 80's that He-Ne laser radiation alters
the firing pattern of nerves; it was also found that transcutaneous
irradiation with HeNe laser mimicked the effect of peripheral
stimulation of a behavioral reflex. These findings were found to be
connected with pain therapy (review [4]).
So,
what happens when the molecule of photoacceptor absorbs photons?
Answer - electronic excitation followed by photochemical reactions
occurring from lower excitation states (first singlet and triplet). It
is also known that electronic excitation of absorbing centers alters
their redox properties. Until yet, five primary reactions have been
discussed in literature (Fig. 4). Two of them are connected with
alteration of redox properties and two mechanisms involve generation
of reactive oxygen species (ROE). Also, induction of local transient
(very short time) heating of absorbing chromophors is possible.
Details of these mechanisms can be found in [4, 5].
There
is no ground to believe that only one of the reactions shown in Fig. 4
occurs when a cell is irradiated and excited electronic states are
promoted. The question is, which mechanism is decisive. It is not
excluded that all mechanisms shown in Fig. 4 lead to a similar result,
to a modulation of redox state of the mitochondria (a shift to more
oxidized direction). However, depending on the light dose and
intensity used, some mechanism(s) can prevail significantly [5].
The
next question is, the following if photoacceptors are located in the
mitochondria, then how the primary reactions occurring under
irradiation in the respiratory chain (Fig. 4) are connected with
DNA and RNA synthesis in the nucleus (the action spectra in Fig. 2A,
B) or with changes in plasma membrane (Fig. 2C)? The principal answer
is that between these events there are secondary (dark) reactions
(cellular signaling cascades or photosignal transduction and
amplification, Fig. 5).
Three
regulation pathways are suggested in Fig. 4. The first one is the
control of photoacceptor over the level of intracellular ATP. It is
known tat even small changes in ATP level can alter cellular
metabolism significantly. This regulation way is especially important
by irradiation of hypoxic, starving or otherways stressed cells.
However, in many cases the regulative role of redox homeostasis is
proved to be more important than that of ATP. For example, it is known
that the susceptibility of cells to hypoxic injury depends more on the
capacity of cells to maintain the redox homeostasis and less on their
capacity to maintain the energy status.
The
second and third regulation pathways are mediated through the cellular
redox state (Eh; Fig. 4). This way involve redox-sensitive
transcription factors (NF-KB and AP1, Fig. 4) or cellular signaling
homeostatic cascades from cytoplasma via cells membrane to the nucleus
(Fig. 4). As a whole, the scheme in Fig. 4 suggests a shift in
overcell redox potential into more oxidized direction. Modulation of
cellular redox state affects gene expression namely via transcription
factors. It is important that in spite of some similar or even
identical steps in cellular signaling, the final cellular responses to
the irradiation differ due to existence of different modes of
regulation of transcription factors. The mechanisms of regulation are
not understood well yet.
The
magnitude of cellular responses depends on cellular redox potential
(and its physiological status, respectively) at the moment of
irradiation. The cellular response is stronger when the redox
potential of the target cell is initially shifted to a more reduced
state (and intracellular pH, pH;, is lowered, as usually happens in
injured cells). This explains why the degrees of cellular responses
can differ markedly in different experiments or in different clinical
cases, and why the effects are sometimes nonexistent.
One
should emphasize that some biological limitations exist for
photobiomodulation effects. These are discussed in [5].
4.
Enhancement of cellular metabolism via activation of nonmitochondrial
photoacceptors. Indirect activation/suppression
The
redox regulation mechanism cannot occur solely via respiratory chain
(Section 3). Other redox chains containing molecules, which absorb
light in visible-to-near IR radiation, and are some key structures
that can regulate a metabolic pathway, can be photoacceptors for
photobiomodulation as well. One such example is NADPH-oxidase of
phagocytic cells, which is responsible for nonmitochondrial
respiratory burst. This multicomponent enzyme system located in the
plasma membrane is a redox chain that generates reactive oxygen
species (ROS) as a response to the microbicidal or other types of
activation. Irradiation with He-Ne laser and diode lasers and LED's
can activate this chain in various phagocytic cells. Many worked
examples can be found in [5]. In phagocytes, the activation of
respiratory chains in mitochondria occurs as well, as NADHP-oxidase
activation, but the latter is much stronger.
ROS,
burst of which is induced by direct irradiation of phagocytes, can
activate or inactivate other cells, which were not irradiated
directly. In this way, indirect activation or suppression of metabolic
pathways in non-irradiated cells occurs. Also, lymphokines and
cytokines produced by irradiated lymphocytes can influence metabolism
of other cells. This situation is common by irradiation on tissues.
5.
Concluding Remarks
The
photobiological action mechanism via activation of respiratory chain
is a universal working mechanism for various cells. Crucial events of
this type of cell metabolism activation are occurring due to a shift
of cellular redox potential into more oxidized direction as well as
due to ATP extrasynthesis. Susceptibility to irradiation and
capability for activation depend on physiological status of irradiated
cells: the cells, which overall redox potential is shifted to more
reduced state (example: some pathological conditions) are more
sensitive to the irradiation. The specificity of final photobiological
response is determined not at the level of primary reactions in the
respiratory chain but at the transcription level during cellular
signaling cascades. In some cells, only partial activation of cell
metabolism happens by this mechanism (example: redox priming of
lymphocytes).
All
light-induced biological effects depend on the parameters of the
irradiation (wavelength, dose, intensity, irradiation time, and
continuous wave or pulsed mode, pulse parameters). According to action
spectra, optimal wavelengths are 820-830, 760, 680, and 620 nn. Large
volumes and deeper layers of tissues can successfully irradiated by
laser only (e.g. inner and middle ear diseases, injured siatic or
optical nerves, deep inflammations etc.). The LED's are excellent for
irradiation of surface injuries.
Cited Literature
1.
Tuner, J. and Hode, L. (1999). Low Level Laser Therapy. Clinical
Practice and Scientific Background. Prima Books, Grangesberg
(Sweden).
2.
Baxter, G.D. (1994). Therapeutic Lasers. Theory and Practice.
Churchill Livingstone, London.
3.
Simunovic, Z., editor (2000). Lasers in Medicine and Dentistry, vol.
I. Vitgraf, Rijeka (Croatia).
4.
Karu, T.I. (2002). Low power laser therapy. In: CRC Biomedical
Photonics Handbook, T. Vo-Dinh, Editor- in-Chief, CRC Press, Boca
Raton (USA).
5.
Karu, T.I. (1998). The Science of Low Power Laser Therapy. Gordon
and Breach Sci. Publ., London.
