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ATP NADH
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Cytochrome |
BIOPHYSICAL ASPECTS
OF LOW LEVEL LASER THERAPY
Herbert Klima Atomic Institute of the Austrian
Universities, Vienna, Austria
Biophysical aspects
of low level laser therapy will be discussed from
two points of view: from the electromagnetic and the
thermodynamical point of view. From electromagnetic
point of view, living systems are mainly governed by
he electromagnetic interaction whose interacting
particles are called photons. Each interaction
beween molecules, macromolecules or living cells is
basically electromagnetic and governed by photons.
For this reason, we must expect that electromagnetic
influences like laser light of proper wavelength
will have remarkable impact on the regulation of
living processes. An impressive example of this
regulating function of various wavelengths of light
is found in the realm of botany, where photons of
660 nm are able to trigger the growth of plants
which leads among other things to the formation of
buds. On the other hand, irradiation of plants by
730 nm photons may stopp the growth and the
flowering. Human phagocyting cells are natively
emitting light which can be detected by single
photon counting methods. Singlet oxygen molecules
are the main sources of this light emitted at 480,
570, 633, 760, 1060 and 1270 nm wavelengths. On the
other hand, human cells (leukocytes, lymphocytes,
stem cells, fibroblasts, etc) can be stimulated by
low power laser light of just these wavelengths.
From thermodynamical
point of view, living systems - in contrast to dead
organisms - are open systems which need metabolism
in order to maintain their highly ordered state of
life. Such states can only exist far from
thermodynamical equilibrium thus dissipating heat in
order to maintain their high order and complexity.
Such nonequilibrium systems are called dissipative
structures proposed by the Nobel laureat I.
Prigogine. One of the main feature of dissipative
structures is their ability to react very sensibly
on weak influences, e.g. they are able to amplify
even very small stimuli. Therefore, we must expect
that even weak laser light of proper wavelength and
proper irradiation should be able to influence the
dynamics of regulation in living systems. For
example, the transition from a cell at rest to a
dividing one will occur during a phase transition
allready influenced by the tinest fluctuations.
External stimuli can induce these phase transitions
which would otherwise not even take place. These
phase transitions induced by light can be
impressively illustrated by various chemical and
physiological reactions as special kinds of
dissipative systems.
One of he most
important biochemical reaction localized in
mitochondria is the oxidation of NADH in the
respiatory chain of aerobic cells. A similar
reaction has been found to be a dissipative process
showing oscillating and chaotic behaviour capable to
absorb and amplify photons of proper wavelength. A
great variety of experimental and clinical results
in the field of low level laser therapy supports
these two biophysical points of view concerning the
interaction beween life and laser light. Our former,
but also our recent experimental results on the
effects of low level laser light on human cells are
steps in this direction. By using cytometric,
photometric and radiochemical methods it is shown
that the increase or decrease of cells growth
depends on the applied wavelenghts (480, 570, 633,
700, 760, 904, 1060, 1270 nm), on the irradiance
(100 - 5000 J/m2), on the pulse sequence modulated
to laser beams (constant, periodic, chaotic pulses),
on the type of cells (leukocytes, lymphocytes,
fibroblasts, normal and cancer cells) and on the
density of the cells in tissue cultures.
Our experimental results support our hypothesis
which states that triplet oxygen molecules are able
to absorb proper laser light at wavelenght at
wavelenghts 480, 570, 633, 700, 760, 904, 1060, 1270
nm thus producing singlet oxygen molecules. Singlet
oxygen takes part in many metabolic processes, e.g.
catalytic oxydation of NADH which has been shown to
be a dissipative system far from thermodynamical
equilibrium and sensitive even to small stimuli.
Therfore, laser light of proper wavelenght and
irradiance in low level laser therapy is assumed to
be able to exicte oxygen molecules thus influencing
or amplifying metabolism and consequently
influencing and supporting fundamental healing
processes
MECHANISMS OF
LOW-POWER LASER LIGHT ACTION ON CELLULAR LEVEL
Tiina Karu Institute
of Laser and Informatic Technologies of Russian
Acad. Sci., 142092 Troitsk, Moscow Region, Russian
Federation
Cytochrome c oxidase is discussed as a possible
photoacceptor when cells are irradiated with
monochromatic red to near-IR radiation. Four primary
action mechanisms are reviewed: changes in the redox
properties of the respiratory chain components
following photoexcitation of their electronic
states, generation of singlet oxygen, localized
transient heating of absorbing chromophores, and
increased superoxide anion production with
subsequent increase in concentration of the product
of its dismutation, H2O2. A cascade of reactions
connected with alteration in cellular homeostasis
parameters (pHi, [Cai], cAMP, Eh, [ATP] and some
others) is considered as a photosignal transduction
and amplification chain in a cell (secondary
mechanisms).
Effect of low-intensity
(3.75-25 J/cm2) near-infrared (810 nm) laser
radiation on red blood cell ATPase
activities and membrane structure
Kujawa J; Zavodnik L; Zavodnik I; Buko V; Lapshyna A; Bryszewska
M
Journal of clinical laser medicine &
surgery; VOL: 22 (2); p. 111-7 /200404/
Department of Rehabilitation, Medical
University of Lodz, Lodz, Poland. jkujawa@bow43.gnet.pl
OBJECTIVE: The biostimulation and therapeutic
effects of low-power laser radiation of different
wavelengths and light doses are well known, but the
exact mechanism of action of the laser radiation
with living cells is not yet understood. The aim of
the present work was to investigate the effect of
laser radiation (810 nm, radiant exposure 3.75-25
J/cm(2)) on the structure of protein and lipid
components of red blood cell membranes and it
functional properties. The role of membrane ATPases
as possible targets of laser irradiation was
analyzed.
BACKGROUND DATA: A variety of studies both in vivo
and in vitro showed significant influence of laser
irradiation on cell functional state. At the same
time another group of works found no detectable
effects of light exposure. Some different
explanations based on the light absorption by
primary endogenous chromophores (mitochondrial
enzymes, cytochromes, flavins, porphyrins) have been
proposed to describe biological effects of laser
light. It was suggested that optimization of the
structural-functional organization of the
erythrocyte membrane as a result of laser
irradiation may be the basis for improving the
cardiac function in patients under a course of laser
therapy. MATERIALS AND METHODS: Human red blood
cells or isolated cell membranes were irradiated
with low-intensity laser light (810 nm) at different
radiant exposures (3.75-25 J/cm(2)) and light powers
(fluence rate; 10-400 mW) at 37 degrees C. As the
parameters characterizing the structural and
functional changes of cell membranes the activities
of Na(+)-, K(+)-, and Mg(2+)-ATPases, tryptophan
fluorescence of membrane proteins and fluorescence
of pyrene incorporated into membrane lipid bilayer
were used. RESULTS: It was found that near-infrared
low-intensity laser radiation changes the ATPase
activities of the membrane ion pumps in the dose-
and fluence rate-dependent manner. At the same time
no changes of such integral parameters as cell
stability, membrane lipid peroxidation level,
intracellular reduced glutathione or oxyhaemoglobin
level were observed. At laser power of 10 mW, an
increase of the ATPase activity was observed with
maximal effect at 12-15 J/cm(2) of light dose
(18-26% for the total ATPase activity). At laser
power of 400 mW (fluence rate significantly
increased), inhibition of ATPases activities mainly
due to the inhibition of Na(+)-, K(+)-ATPase was
observed with maximal effect at the same light dose
of 12-15 J/cm(2) (18-23% for the total ATPase
activity).
Fractionation of the light dose significantly
changed the membrane response to laser radiation.
Changes in tryptophan fluorescent parameters of
erythrocyte membrane proteins and the increase in
lipid bilayer fluidity measured by pyrene monomer/excimer
fluorescence ratio were observed.
CONCLUSIONS: Near-infrared laser light radiation
(810 nm) induced long-term conformational
transitions of red blood cell membrane which were
related to the changes in the structural states of
both erythrocyte membrane proteins and lipid bilayer
and which manifested themselves as changes in
fluorescent parameters of erythrocyte membranes and
lipid bilayer fluidity. This resulted in the
modulation of membrane functional properties:
changes in the activity of membrane ion pumps and,
thus, changes in membrane ion flows.
Cellular effects of low power laser
therapy can be mediated by nitric oxide.
Karu TI; Pyatibrat LV; Afanasyeva NI
Lasers in surgery and medicine; VOL: 36 (4);
p. 307-14 /200504/
Institute of Laser and Information Technologies of
the Russian Academy of Sciences, 142190 Troitsk,
Moscow, Russia. tkaru@isan.troitsk.ru
BACKGROUND AND OBJECTIVES: The objective of this
study was to investigate the possibility of
involvement of nitric oxide (NO) into the
irradiation-induced increase of cell attachment.
These experiments were performed with a view to
exploring the cellular mechanisms of low-power laser
therapy. STUDY DESIGN/MATERIALS AND METHODS: A
suspension of HeLa cells was irradiated with a
monochromatic visible-to-near infrared radiation
(600-860 nm, 52 J/m2) or with a diode laser (820 nm,
8-120 J/m2) and the number of cells attached to a
glass matrix was counted after 30 minute incubation
at 37 degrees C. The NO donors sodium nitroprusside
(SNP), glyceryl trinitrate (GTN), or sodium nitrite
(NaNO2) in the concentration range 5 x 10(-9)-5 x
10(-4)M were added to the cellular suspension before
or after irradiation. The action spectra and the
concentration and fluence dependencies obtained were
compared and analyzed.
RESULTS: The well-structured action spectrum for the
increase of the adhesion of the cells, with maxima
at 619, 657, 675, 740, 760, and 820 nm, points to
the existence of a photoacceptor responsible for the
enhancement of this property (supposedly cytochrome
c oxidase, the terminal respiratory chain enzyme),
as well as signaling pathways between the cell
mitochondria, plasma membrane, and nucleus. Treating
the cellular suspension with SNP (5 x 10(-5)M)
before irradiation significantly modifies the action
spectrum for the enhancement of the cell attachment
property (band maxima at 642, 685, 700, 742, 842,
and 856 nm). The action of SNP, GTN, and NaNO2 added
before or after irradiation depends on their
concentration and radiation fluence.
CONCLUSIONS: The NO donors added to the cellular
suspension before irradiation eliminate the
radiation-induced increase in the number of cells
attached to the glass matrix, supposedly by way of
binding NO to cytochrome c oxidase. NO added to the
suspension after irradiation can also inhibit the
light-induced signal downstream. Both effects of NO
depend on the concentration of the NO donors added.
These results indicate that NO can control the
irradiation-activated reactions that increase the
attachment of cells.
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