Laser therapy ATP, NO, cellular effects

Effect of pre-irradiation with different doses, wavelengths, and application intervals of low-level laser therapy on cytochrome c oxidase activity in intact skeletal muscle of rats


Modulation of cytochrome c oxidase activity has been pointed as a possible key mechanism for low-level laser therapy (LLLT) in unhealthy biological tissues. But recent studies by our research group with LLLT in healthy muscles before exercise found delayed skeletal muscle fatigue development and improved biochemical status in muscle tissue. Therefore, the aim of this study was to evaluate effects of different LLLT doses and wavelengths in cytochrome c oxidase activity in intact skeletal muscle. In this animal experiment, we irradiated the tibialis anterior muscle of rats with three different LLLT doses (1, 3, and 10 J) and wavelengths (660, 830, and 905 nm) with 50 mW power output. After irradiation, the analyses of cytochrome c oxidase expression by immunohistochemistry were analyzed at 5, 10, 30 min and at 1, 2, 12, and 24 h.

Our results show that LLLT increased (p < 0.05) cytochrome c oxidase expression mainly with the following wavelengths and doses: 660 nm with 1 J, 830 nm with 3 J, and 905 nm with 1 J at all time points. We conclude that LLLT can increase cytochrome c oxidase activity in intact skeletal muscle and that it contributes to our understanding of how LLLT can enhance performance and protect skeletal muscles against fatigue development and tissue damage. Our findings also lead us to think that the combined use of different wavelengths at the same time can enhance LLLT effects in skeletal muscle performance and other conditions, and it can represent a therapeutic advantage in clinical settings.


Photomedicine and Laser Surgery, Volume 31, Number 2, 2013

Low-Intensity Laser Irradiation at 660nm Stimulates Transcription of Genes Involved in the Electron Transport Chain


Background data:Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I�IV) and oxidative phosphorylation (ATP synthase).

Methods: Four human skin fibroblast cell models were used in this study, normal nonirradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660nm diode laser with a fluence of 5 J/cm2 and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). Results: LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+ transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K + exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells.

Conclusions: LILI at 660nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

Photomedicine and Laser Surgery. August 2012, 30(8): 451-459.

Effect of 635nm Light-Emitting Diode Irradiation on Intracellular Superoxide Anion Scavenging Independent of the Cellular Enzymatic Antioxidant System

To cite this article:
WonBong Lim, JiSun Kim, ChaeGwang Lim, SangWoo Kim, SangMi Jeon, Sandeep Karna, MinSung Cho, HongRan Choi, and OkJoon Kim. Photomedicine and Laser Surgery. August 2012, 30(8): 451-459. doi:10.1089/pho.2011.3199.


Objective: The aim of this study was to examine the reactive oxygen species (ROS) that are dissipated by 635nm irradiation, and the effect of 635nm irradiation on ROS scavenging system.

Background data: Intracellular ROS are produced in the form of superoxide anion by either nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase in response to a number of stimuli. Low-level light irradiation decreases the intracellular ROS level and has been used in clinical situations for reducing the level of oxidative stress.

Methods: Human epithelial cells were exposed to exogenous and endogenous oxidizing agents that promote the generation of harmful ROS. These were then irradiated with 635�nm LED light, 5 mW/cm2 for 1�h, 18�J/cm2 or by 470�nm LED light, also 5 mW/cm2 for 1�h, 18�J/cm2 on a 9�cm cell culture dish. After irradiation, the MTT reduction method and malondialdehyde (MDA) colorimetric assay were performed in xanthine/xanthine oxidase (XXO)- or hydrogen peroxide (H2O2)-treated HaCaT cells. The superoxide anion was detected by an electron spin resonance (ESR) spectrometer using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap and H2O2 was assayed by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA).

Results: Irradiation at 635�nm enhanced cell viability in the XXO-treated HaCaT cells. Also, irradiation had a much lesser effect on cell viability in the HaCaT cells treated with exogenous H2O2 as compared with that in cells treated with N-acetyl-L-cysteine. The level of the superoxide anion increased in response to XXO treatment, and then decreased after 635�nm irradiation. Irradiation with 635�nm led to a decrease in superoxide anion and lipid peroxidation levels in the presence or absence of diethyldithiocarbamate.

Conclusions: These results highlight the potential role of 635�nm irradiation in protection against oxidative stress by scavenging superoxide anions. Also, a pathway that is independent of the activities of intracellular enzymatic ROS scavengers, such as superoxide dismutase, glutathione peroxidase and catalase might be involved in its mechanism of action.

Laser Photobiomodulation of Gene Expression and Release of Growth Factors and Cytokines from Cells in Culture:

A Review of Human and Animal Studies


Philip V. Peplow, Ph.D.,1 Tzu-Yun Chung, BMedSc,1 Brigid Ryan, B.A.,2 and G. David Baxter, DPhil2


Aim: The aim of this paper was to review experimental studies of laser irradiation of human and animal cells in culture to assess the photobiomodulatory effects of such irradiation on gene expression and release of growth factors and cytokines. Background: Previous studies have reported that the release of certain growth factors and cytokines by various types of cells can be modulated by laser irradiation. A review of recent studies may provide further insight into the clinical effects brought about by laser irradiation of cells and tissues, including changes in inflammation and cellular proliferation.

Methods: A systematic review was completed of original research papers investigating the effects of laser phototherapy on gene expression and release of these mediators from human and animal cells in culture ( January 2002 to September 2009). Relevant papers were retrieved primarily from PubMed and Medline databases. The search terms were ��growth factors��, ��cytokines��, ��laser therapy��, ��laser irradiation��, ��laser phototherapy,�� and ��phototherapy��. Results: A total of 17 relevant papers were included in the review, comprising studies on a variety of cell types. Considerable variation occurred in research design, methodology, and irradiation parameters employed, limiting comparison of research findings between studies. Results demonstrated that laser irradiation at green, red, or infrared wavelengths at a range of dosage parameters can cause significant changes in the cellular gene expression and release of these mediators, and that such effects depend upon wavelength and radiant exposure.

Conclusions: Findings from the reviewed studies clearly demonstrate the ability of laser irradiation to modulate gene expression and the release of growth factors and cytokines from cells in culture. Further work is indicated at the clinical level to elucidate the putative clinical relevance of these cellular-level effects.

Intracellular ATP Level Increases in Lymphocytes Irradiated with Infrared Laser Light of Wavelength 904 nm

Stefano Benedicenti, D.D.S.,1 Isidoro Mario Pepe,1 Francesca Angiero, M.D.,2 and Alberico Benedicenti, D.D.S.1


Objective: Red and near-infrared laser irradiation is reported to have a range of biological effects on cultured cells and different tissues, leading to the hypothesis that laser light can affect energy metabolism. Increased adenosine triphosphate (ATP) synthesis has been reported in cultured cells and rat brain tissue after irradiation at 632.8 nm and 830 nm, respectively. This study investigated whether diode pulsed laser irradiation enhances ATP production in lymphocytes.

Materials and Methods: Aliquots (500 L) of an extract of cultured lymphocytes of the Molt-4 cell line were irradiated with diode laser light ( 904 nm, pulsed mode, 6 kHz frequency) with an average emission power of 10 mW for 60 min. A Spectra Physics M404 power meter was used to measure light intensity. Controls were treated similarly but not irradiated. The amount of ATP was measured by the luciferin-luciferase bioluminescent assay.

Results: The amount of ATP in irradiated cell cultures was 10.79  0.15 g/L (SD; n 10), and in non-irradiated cell cultures it was 8.81 0.13 g/L (SD; n 10). The average percentage increase of irradiated versus control cell cultures was about 22.4%  0.56% SD (p 0.001).

Conclusions: This significant increase is probably due to laser irradiation; it cannot be attributed to any thermal effect, as the temperature during irradiation was maintained at 37.0°  0.5°C. Thus the therapeutic effects of the biostimulating power of this type of laser are identified and its indications may be expanded.

This email address is being protected from spambots. You need JavaScript enabled to view it.

Ga-As (808 nm) Laser Irradiation Enhances ATP Production in Human Neuronal Cells in Culture

Jun 2007, Vol. 25, No. 3 : 180 -182

U. Oron, Ph.D.
Photothera Inc., Carlsbad, California.

S. Ilic, M.D.
Photothera Inc., Carlsbad, California.

L. De Taboada, M.S.E.E.
Photothera Inc., Carlsbad, California.

J. Streeter, M.D.
Photothera Inc., Carlsbad, California.

Objective: The aim of the present study was to investigate whether Ga-As laser irradiation can enhance adenosine triphosphate (ATP) production in normal human neural progenitor (NHNP) cells in culture. Methods: NHNP were grown in tissue culture and were treated by Ga-As laser (808 nm, 50 mW/cm2, 0.05 J/cm2), and ATP was determined at 10 min after laser application.

Results: The quantity of ATP in laser-treated cells was 7513 ± 970 units, which was significantly higher (p < 0.05) than the non-treated cells, which comprised 3808 ± 539 ATP units.

Conclusions: Laser application to NHNP cells significantly increases ATP production in these cells. These findings may explain the beneficial effects of low-level laser therapy (LLLT) in stroked rats. Tissue culture of NHNP cells might offer a good model to study the mechanisms associated with promotion of ATP production in the nervous system by LLLT.

Photochemistry and Photobiology of Light Absorption by Living Cells

Apr 2006, Vol. 24, No. 2: 179-185 Photomedicine and Laser Surgery

Dr. Rachel Lubart, Ph.D.
Department of Physics, Bar-Ilan University, Ramat-Gan, Israel.

Ronit Lavi, Ph.D.
Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel.

Harry Friedmann, Ph.D.
Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel.

Shimon Rochkind, M.D.
Division of Peripheral Nerve Reconstruction, Tel-Aviv Sourasky Medical Center, Tel-Aviv University, Tel-Aviv, Israel.

Objective: In this review, we summarize a part of our research concerning photobiostimulative effects on cardiomyocytes, sperm cells, and nerve cells. We concentrate on results demonstrating that photobiostimulation can be described by the Arndt-Schultz (A.S.) curve.

Results: monitoring an increase in reactive oxygen species (ROS) concentration following visible light irradiation describe the ascending part of the A.S. curve, whereas those that describe the antioxidant role of photobiostimulation represent the descending part of the curve.

This email address is being protected from spambots. You need JavaScript enabled to view it.

Photoengineering of Tissue Repair in Skeletal and Cardiac Muscles

Apr 2006, Vol. 24, No. 2: 111-120, Photomedicine and Laser Surgery

Dr. Uri Oron, Ph.D.
Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel.

This review discusses the application of He-Ne laser irradiation to injured muscles at optimal power densities and optimal timing, which was found to significantly enhance (twofold) muscle regeneration in rats and, even more, in the cold-blooded toads. Multiple and frequent (daily) application of the laser in the toad model was found to be less effective than irradiation on alternate days. It was found that in the ischemia/reperfusion type of injury in the skeletal leg muscles (3 h of ischemia), infrared Ga-Al-As laser irradiation reduced muscle degeneration, increased the cytoprotective heat shock proteins (HSP-70i) content, and produced a twofold increase in total antioxidants. In vitro studies on myogenic satellite cells (SC) revealed that phototherapy restored their proliferation. Phototherapy induced mitogen-activated protein kinase/extracellular signalregulated protein kinase (MAPK/ERK) phosphorylation in these cells, probably by specific receptor phosphorylation. Cell cycle entry and the accumulation of satellite cells around isolated single myofibers cultured in vitro was also stimulated by phototherapy. Phototherapy also had beneficial effects on mouse, rat, dog and pig ischemic heart models. In these models, it was found that phototherapy markedly and significantly reduced (50�70%) the scar tissue formed after induction of myocardial infarction (MI). The phototherapeutic effect was associated with reduction of ventricular dilatation, preservation of mitochondria and elevation of HSP- 70i and ATP in the infarcted zone.

This email address is being protected from spambots. You need JavaScript enabled to view it.


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.

Amat A, Rigau J, Nicolaua R, Aalders M et al.

Effect of red and near-infrared laser light on adenosine triphosphate (ATP) in the luciferin luciferase reaction.

Journal of Photochemistry and Photobiology A: Chemistry. 2004; 168 (1-2): 59-65.

Adenosine triphosphate (ATP) is an important molecule in biology because it stores chemical energy and releases it to the biochemical processes occurring in the cell. In this study the authors analysed the biochemical behaviour of ATP after irradiating it with 635 and 830 nm diode lasers. They analysed the luminescence peak, the reaction rate and the area under the luminescence curve at 2�10�9 mol/l of ATP in the luciferine�luciferase luminescence reaction before and after irradiating the molecule at several irradiances and radiant exposures. The absorption spectrum of ATP at 3�10�3mol/l concentration was measured between 650 and 900 nm after laser irradiation at 635 nm (Argon-Dye) and 830 nm (diode laser). It was found significant differences in the measured parameters when ATP was irradiated with both wavelengths. The absorption spectra of non-irradiated and irradiated ATP showed a physical�chemical difference in the ATP molecule after irradiation with both lasers.It can concluded that visible and near-IR laser light with the parameters that were used in this study changed the biochemical behaviour of ATP molecules.


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. This email address is being protected from spambots. You need JavaScript enabled to view it.

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. This email address is being protected from spambots. You need JavaScript enabled to view it.

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.



Fabrikstr. 22
79183 Waldkirch