Cellular Studies

Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy.

Silveira PC, Streck EL, Pinho RA.

J Photochem Photobiol B. 2006 Nov 17

Laboratorio de Fisiologia e Bioquimica do Exercicio, Universidade do Extremo Sul Catarinense, 88806-000 Criciuma, SC, Brazil.

Laser therapy is used in many biomedical sciences to promote tissue regeneration. Many studies involving low-level laser therapy have shown that the healing process is enhanced by such therapy. In this work, we evaluated mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase activities in wounds after irradiation with low-level laser. The animals were divided into two groups: group 1, the animals had no local nor systemic treatment and were considered as control wounds; group 2, the wounds were treated immediately after they were made and every day after with a low-level laser (AsGa, wavelength of 904nm) for 10 days. The results showed that low-level laser therapy improved wound healing. Besides, our results showed that low-level laser therapy significantly increased the activities of complexes II and IV but did not affect succinate dehydrogenase activity. These findings are in accordance to other works, where cytochrome c oxidase (complex IV) seems to be activated by low-level laser therapy. Besides, we showed, for the first time, that complex II activity was also activated. More studies are being carried out in order to evaluate other mitochondrial enzymes activities after different doses and irradiation time of low-level laser.

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The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation.

Hawkins DH, Abrahamse H.

Lasers Surg Med. 2006 Jan;38(1):74-83.

Faculty of Health, University of Johannesburg, P.O. Box 17011, Doornfontein, Johannesburg 2028, South Africa.

BACKGROUND: In medicine, lasers have been used predominantly for applications, which are broadly termed low level laser therapy (LLLT), phototherapy or photobiomodulation. This study aimed to establish cellular responses to Helium-Neon (632.8 nm) laser irradiation using different laser fluences (0.5, 2.5, 5, 10, and 16 J/cm(2)) with a single exposure on 2 consecutive days on normal and wounded human skin fibroblasts.

MATERIALS AND METHODS: Changes in normal and wounded fibroblast cell morphology were evaluated by light microscopy. Changes following laser irradiation were evaluated by assessing the mitochondrial activity using adenosine triphosphate (ATP) luminescence, cell proliferation using neutral red and an alkaline phosphatase (ALP) activity assay, membrane integrity using lactate dehydrogenase (LDH), and percentage cytotoxicity and DNA damage using the Comet assay.

RESULTS: Morphologically, wounded cells exposed to 5 J/cm(2) migrate rapidly across the wound margin indicating a stimulatory or positive influence of phototherapy. A dose of 5 J/cm(2) has a stimulatory influence on wounded fibroblasts with an increase in cell proliferation and cell viability without adversely increasing the amount of cellular and molecular damage. Higher doses (10 and 16 J/cm(2)) were characterized by a decrease in cell viability and cell proliferation with a significant amount of damage to the cell membrane and DNA.

CONCLUSIONS: Results show that 5 J/cm(2) stimulates mitochondrial activity, which leads to normalization of cell function and ultimately stimulates cell proliferation and migration of wounded fibroblasts to accelerate wound closure. Laser irradiation can modify cellular processes in a dose or fluence (J/cm(2)) dependent manner.

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Gavish L, Asher Y, Becker Y, Kleinman Y.

Lasers Surg Med. 2004;35(5):369-76.

Department of Molecular Virology, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.

BACKGROUND AND OBJECTIVES: Low level laser irradiation (LLLI) is used to promote wound healing. Molecularly it is known to stimulate mitochondrial membrane potential (MMP), cytokine secretion, and cell proliferation. This study was designed to determine the influence of LLLI on the kinetics of MMP stimulation and decay, specific cytokine gene expression, and subcellular localization of promyelocytic leukemia (PML) protein on HaCaT human keratinocytes.

STUDY DESIGN/MATERIAL AND METHODS: The cells were irradiated by a 780 nm titanium-sapphire (Ti-Sa) laser with 2 J/cm(2) energy density. MMP was monitored with Mitotracker, a mitochondrial voltage-sensitive fluorescent dye. Cytokine gene expression was carried out using semi-quantitative-reverse transcription polymerase chain reaction. Subcellular localization of PML protein, a cell-cycle checkpoint protein, was determined using immunofluorescent staining.

RESULTS: The fluorescence intensity of MMP was increased immediately after the end of LLLI by 148 +/- 6% over control (P<0.001). Subsequently it decayed, reaching 51 +/- 14% of the control level (P < 0.01) within 200 minutes. This decay was characterized by an exponential curve (R = 0.96) with a lifetime of 79 +/- 36 minutes (P < 0.05). Following irradiation, the expression of interleukin-1alpha, interleukin-6, and keratinocyte growth factor (KGF) genes were transiently upregulated; but the expression of the proinflammatory gene interleukin-1beta, was suppressed. The subnuclear distribution of PML was altered from discrete domains to its dispersed form within less than 1 hour after LLLI.

CONCLUSIONS: These changes reflect a biostimulative boost that causes a shift of the cell from a quiescent to an activated stage in the cell cycle heralding proliferation and suppression of inflammation. Further characterization of MMP kinetics may provide a quantitative basis for assessment of the effect of LLLI in the clinical setting

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Vinck EM, Cagnie BJ, Cornelissen MJ, Declercq HA, Cambier DC.

Lasers Med Sci. 2003;18(2):95-9.

Department of Rehabilitation Sciences and Physiotherapy, Ghent University, 9000 Ghent, Belgium.

BACKGROUND AND OBJECTIVE: As Light Emitting Diode (LED) devices are commercially introduced as an alternative for Low Level Laser (LLL) Therapy, the ability of LED in influencing wound healing processes at cellular level was examined.

STUDY DESIGN/MATERIALS AND METHODS: Cultured fibroblasts were treated in a controlled, randomized manner, during three consecutive days, either with an infrared LLL or with a LED light source emitting several wavelengths (950 nm, 660 nm and 570 nm) and respective power outputs. Treatment duration varied in relation to varying surface energy densities (radiant exposures).

RESULTS: Statistical analysis revealed a higher rate of proliferation (p < 0.001) in all irradiated cultures in comparison with the controls. Green light yielded a significantly higher number of cells, than red (p < 0.001) and infrared LED light (p < 0.001) and than the cultures irradiated with the LLL (p < 0.001); the red probe provided a higher increase (p < 0.001) than the infrared LED probe and than the LLL source.

CONCLUSION: LED and LLL irradiation resulted in an increased fibroblast proliferation in vitro. This study therefore postulates possible stimulatory effects on wound healing in vivo at the applied dosimetric parameters.

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Medrado AR, Pugliese LS, Reis SR, Andrade ZA.

Lasers Surg Med. 2003;32(3):239-44.

Laboratory of Experimental Pathology, Oswaldo Cruz Foundation-Salvador, Bahia, Brazil.

BACKGROUND AND OBJECTIVE: In re-evaluating the effects of laser therapy in wound healing, the role of extracellular matrix elements and myofibroblasts, was analyzed.

STUDY DESIGN/MATERIALS AND METHODS: Cutaneous wounds were inflicted on the back of 72 Wistar rats. Low level laser was locally applied with different energy densities. Lesions were analyzed after 24, 48, 72 hours and 5, 7, and 14 days. Tissues were studied by histology, immunohistochemistry, and electron microscopy.

RESULTS: In treated animals, the extent of edema and the number of inflammatory cells were reduced (P < 0.05), but the amount of collagen and elastic fibers appeared slightly increased. Desmin/smooth muscle alpha-actin-phenotype myofibroblasts were statistically more prominent on the 3rd day after surgery (P < 0.05) in treated wounds than in controls. Treatment with a dosage of 4 J/cm(2) was superior to that with 8 J/cm(2).

CONCLUSIONS: Laser therapy reduced the inflammatory reaction, induced increased collagen deposition and a greater proliferation of myofibroblasts in experimental cutaneous wounds.

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Pereira AN, Eduardo Cde P, Matson E, Marques MM.

Lasers Surg Med. 2002;31(4):263-7.

Department of Stomatology, School of Dentistry, University of Sao Paulo-SP, Brazil 05508-900.

BACKGROUND AND OBJECTIVES: In dentistry, low-power lasers have been used in the treatment of dentin hypersensitivity, gingivitis, periodontitis, and different forms of oral ulcers. This in vitro study focuses on the biostimulation of NIH-3T3 fibroblasts by a low-power Ga-As-pulsed laser.

STUDY DESIGN/MATERIALS AND METHODS: We have studied cell growth and procollagen synthesis of cultured fibroblasts submitted to low-power laser irradiation with energy densities varying from 3 to 5 J/cm(2) over a period of 1-6 days. The light source was a 120 mW Ga-As diode laser (lambda = 904 nm). Growth curves and procollagen immunoprecipitation were obtained.

RESULTS: Irradiation of 3 and 4 J/cm(2) increased the cell numbers about threefold to sixfold comparing to control cultures. However, this effect was restricted to a small range of energy densities since 5 J/cm(2) had no effect on cell growth. The energy density of 3 J/cm(2) remarkably increased cell growth, with no effect on procollagen synthesis, as demonstrated by the immunoprecipitation analysis.

CONCLUSIONS: Our results showed that a particular laser irradiation stimulates fibroblast proliferation, without impairing procollagen synthesi