This PDF file contains the front matter associated with SPIE Proceedings Volume 7165, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

This review will focus on the role of reactive oxygen species in the cellular and tissue effects of low level light therapy (LLLT). Coincidentally with the increase in electron transport and in ATP, there has also been observed by intracellular fluorescent probes and electron spin resonance an increase in intracellular reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, singlet oxygen and hydroxyl radical. ROS scavengers, antioxidants and ROS quenchers block many LLLT processes. It has been proposed that light between 400-500- nm may produce ROS by a photosensitization process involving flavins, while longer wavelengths may directly produce ROS from the mitochondria. Several redox-sensitive transcription factors are known such as NF-kB and AP1, that are able to initiate transcription of genes involved in protective responses to oxidative stress. It may be the case that LLLT can be pro-oxidant in the short-term, but anti-oxidant in the long-term.

Studies in our laboratory demonstrate that the action spectrum for stimulation of cytochrome oxidase activity and cellular ATP parallels the near-infrared absorption spectrum of cytochrome oxidase and that 660-680 nm irradiation upregulates cytochrome oxidase activity in cultured neurons. Treatment with nearinfrared light augments cellular energy production and neuronal viability following mitochondrial injury linking the actions of red to near-infrared light on mitochondrial metabolism in vitro and cell injury in vivo. NIR light treatment represents an innovative therapeutic approach for disease processes in which mitochondrial dysfunction is postulated to play a role including Parkinson's disease, laser eye injury and Age-related macular degeneration.

Following the absorption of photons by cells either resident in or in transit through the skin at and around a wound site, healing can be modulated. This is due to the primary, secondary and tertiary cellular effects of the photons. The main primary effect of phototherapy is photon absorption. This initiates secondary effects within the cells that have absorbed the photons. Secondary effects are restricted to cells that have absorbed a suprathreshold quantity of photonic energy. Photon absorption can lead to an increase in ATP synthesis and the release of reactive oxygen species that can activate specific transcription factors resulting in changes in synthesis of the enzymes needed for cellular proliferation, migration, phagocytosis and protein synthesis, all essential for wound healing. The amount of ATP production is limited in each cell by the availability of ADP and phosphate. Spatial and temporal amplification of the effects of photon absorption increases the range and duration of phototherapy. It may be caused in part by tertiary effects initiated in cells that have not absorbed photons by regulatory proteins such as cytokines secreted by cells that have absorbed photons. Amplification may also be due to changes induced by photons in immune cells, stem cells and soluble protein mediators while in transit through the dermal capillaries. The peripheral location of these capillaries makes their contents readily accessible to photons. The longer the duration of treatment, the greater will be the number of cells in transit that can be affected by photons. Depth of effect may be increased by transduction of electromagnetic energy into mechanical energy. For a treatment to be clinically effective on wound healing, its duration and power may each be important. Components of the immune system, endocrine system and nervous system may also amplify the effects of photons on wound healing.

The importance of coherence in phototherapy has been questioned over the last two decades, with the arguments largely being based on; 1) Lasers are just convenient machines that produce radiation, 2) It is the radiation that produces the photobiological and/or photophysical effects and therapeutic gains, not the machines, and 3) Radiation must be absorbed to produce a chemical or physical change, which results in a biological response. Whilst these conclusions are, in essence, true, they neglect to account for the effects of laser speckle in vivo. In a proportion of individual laser speckles the intensity is higher than the surrounding environment, and the light is partially linearly polarized. This is important because the probability for a photon absorption event to occur largely depends on intensity and the photon absorption cross section of the molecule (which in turn is influenced by polarization and several other factors). In superficial tissue, where the photon flux is high (less absorption has taken place), it is easy to reach necessary power density thresholds without the benefits of laser speckle. However, in deep tissue where the photon flux is extremely low, the increased probability of photon absorption from individual laser speckles increases the probability of reaching the necessary power density thresholds. Because of the non-coherent nature of radiation from light/IR emitting diodes speckle does not occur in the tissue with LED therapy, which may explain why head-to-head comparisons between lasers and LEDs in deep tissue seem to be in favor of lasers, and super-pulsed lasers in particular.

Continuous wave He-Ne laser exposures (Intensity = 35 mW/cm², λ=632.8nm, Fluence range: 1J/cm² to 50 J/cm²) on non-confluent and fully confluent human malignant glioblastoma cells was found to increase the cellular production levels of H₂O₂. Modulations in the cellular metabolic activity were detected (through the MTS assay) three days after the laser irradiation. The metabolic activity was found to be dependent on the laser fluence for both cell growth conditions. Furthermore, three days after the laser exposure, the potential laser induced "bystander" effect was tested through the transfer of growth media from laser irradiated cells onto non-irradiated cells. After two additional days of incubation (5 days post exposure), the non-laser irradiated cells grown under the non-confluent condition were found to have a significant increase in their metabolic activities, whereas minimal to null response was found for the fully confluent condition. For cells grown under the non-confluent conditions, modulations in the metabolic activities in the non-irradiated cells were found to be laser fluence dependent from the initial laser exposed cells treatment conditions. The results herein support the hypothesis of an important role for light enhanced cellular H₂O₂ generation to yield bio-modulatory effects locally and at a distance. The classical "bi-phasic" modulation response of cells to light irradiation is hypothesized to depend upon the quantity of light enhanced H₂O₂ molecules generated from the mitochondria and the number of cells which interact with the H₂O₂ molecules.

Background/purpose: Stress induced premature senescence (SIPS) is defined as the long-term effect of subcytotoxic stress on proliferative cell types. Cells in SIPS display differences at the level of protein expression which affect energy metabolism, defense systems, redox potential, cell morphology and transduction pathways. This study aimed to determine the effect of laser irradiation on second messengers in senescent cells and to establish if that effect can be directly linked to changes in cellular function such as cell viability or proliferation. Materials and Methods: Human keratinocyte cell cultures were modified to induce premature senescence using repeated sub-lethal stresses of 200 uM H₂O₂ or 5% OH every day for four days with two days recovery. SIPS was confirmed by senescence-associated β-galactosidase staining. Control conditions included normal, repeated stress of 500 uM H₂O₂ to induce apoptosis and 200 uM PBN as an anti-oxidant or free radical scavenger. Cells were irradiated with 1.5 J/cm² on day 1 and 4 using a 648 nm diode laser (3.3 mW/cm²) and cellular responses were measured 1 h post irradiation. The affect on second messengers was assessed by measuring cAMP, cGMP, nitric oxide and intracellular calcium (Ca²⁺) while functional changes were assessed using cell morphology, ATP cell viability, LDH membrane integrity and WST-1 cell proliferation. Results: Results indicate an increase in NO and a decrease in cGMP and Ca²⁺ in 200 uM H2O2 irradiated cells while PBN irradiated cells showed a decrease in cAMP and an increase in ATP viability and cell proliferation. Conclusion: Laser irradiation influences cell signaling which ultimately changes the biological function of senescent cells. If laser therapy can stimulate the biological function of senescent cells it may be beneficial to conditions such as immune senescence, skin ageing, muscle atrophy, premature ageing of arteries in patients with advanced heart disease, neurodegenerative disorders and chronic renal failure.

Aim: The aim of the present investigation was to compare, using the MTT Assay, the effects of a polarized light system (λ400-2000nm, 40mW, φ5cm, 9.6J/cm2) on cellular cultures of human laryngeal carcinoma (H.Ep.2) and in a fibroblast lineage (L929-CLLINCTC clone 929). Summary Background Data: Recently there has been increased interest in the propagation of polarized light in randomly scattering media, such as biological tissues, because their high potential of applications, particularly in biomedical area. Materials and Methods: The illuminations were performed at the following times: T0 (24 hours after handling the cells) and T48 (equivalent to 48 hours after the first illumination). The cellular viability was assessed using MTT essay at the following times T0, T6, T12, T24, T48, T72. The results were analyzed using the Graphpad Prism® software. Results: The results showed that time had influenced on the cellular viability of L929 on both control (P=0.0014) and illuminated cultures (P=0.0035). Significant difference between control (P=0.0001) and illuminated H.Ep.2 cells (P=0.0001) was observed. There was a significant difference between the two used types of cells illuminated when compared to their controls: H.Ep.2 (P=0.0001) and L929 (P=0.0002). Conclusion: The use of polarized light on H.Ep.2 and L929 cells resulted on photobiological effects that need further investigation of the mechanisms involved as this is the first study using this methodology.

Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation remain unclear. In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810-nm laser radiation. Significant activation of NFkB was observed for fluences higher than 0.003 J/cm². NF-kB activation by laser was detectable at 1-hour time point. Moreover, we demonstrated that laser phosphorylated both IKK α/β and NF-kB 15 minutes after irradiation, which implied that laser activates NF-kB via phosphorylation of IKK α/β. Suspecting mitochondria as the source of NF-kB activation signaling pathway, we demonstrated that laser increased both intracellular reactive oxygen species (ROS) by fluorescence microscopy with dichlorodihydrofluorescein and ATP synthesis by luciferase assay. Mitochondrial inhibitors, such as antimycin A, rotenone and paraquat increased ROS and NF-kB activation but had no effect on ATP. The ROS quenchers N-acetyl-L-cysteine and ascorbic acid abrogated laser-induced NF-kB and ROS but not ATP. These results suggested that ROS might play an important role in the signaling pathway of laser induced NF-kB activation. However, the western blot showed that antimycin A, a mitochondrial inhibitor, did not activate NF-kB via serine phosphorylation of IKK α/β as the laser did. On the other hand, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that light also upregulates mitochondrial respiration. ATP upregulation reached a maximum at 0.3 J/cm² or higher. We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive transcription factor NF-kB by generating ROS as signaling molecules.

The ability of laser light to modulate specific biological processes has been well documented but the precise mechanism mediating these photobiological interactions remains an area of intense investigation. We recently published the results of our clinical trial with 30 patients in an oral tooth-extraction wound healing model using a 904nm GaAs laser (Oralaser 1010, Oralia, Konstnaz, Germany), assessing healing parameters using routine histopathology and immunostaining (Arany et al Wound Rep Regen 2007, 15, 866). We observed a better organized healing response in laser irradiated oral tissues that correlated with an increased expression of TGF-beta1 immediately post laser irradiation. Our data suggested the source of latent TGF-beta1 might be from the degranulating platelets in the serum, an abundant source of in vivo latent TGF-beta, in the freshly wounded tissues. Further, we also demonstrated the ability of the low power near-infrared laser irradiation to activate the latent TGF-beta complexes in vitro at varying fluences from 10sec (0.1 J/cm²) to 600secs (6 J/cm²). Using serum we observed two isoforms, namely TGF-beta1 and TGF-beta3, were capable of being activated by laser irradiation using an isoform-specific ELISA and a reporter based (p3TP) assay system. We are presently pursuing the precise photomolecular mechanisms focusing on potential chromophores, wavelength and fluence parameters affecting the Latent TGF-beta activation process in serum. As ROS mediated TGF-beta activation has been previously demonstrated and we are also exploring the role of Laser generated-ROS in this activation process. In summary, we present evidence of a potential molecular mechanism for laser photobiomodulation in its ability to activate latent TGF-beta complexes.

Oral Epithelial dysplasia may be a white, red or mixed patch that may affect several sites of the mouth. Chemo-induced precancerous lesions are standard model to study cancer on the oral cavity. The use of Laser photobiomodulation on the oral care is a standard procedure these days and it is known that it proliferative effects on both cells and tissues depending on dose, wavelengths and other parameters. The aim of this study was to assess the effect of laser light on the evolution of chemo-induced epithelial dysplasia on the hamster cheek pouch model. Sixteen animals were divided into four groups: Control (n=8), Laser λ660nm (n=4), and Laser λ790nm (n=4). DMBA induction was carried out three times a week. All animals presented epithelial dysplasia seven days after first induction. When appropriate, laser (λ660nm or λ790nm, 30/40mW, Φ ~ 3mm, 4J/cm2) was used at 48 h interval during two weeks. Chemo-induction continued during all experimental period (6 weeks). Following animal death, specimens were taken, routinely process to wax, cut and stained with H.E. Slides were analyzed under light microscopy by an oral pathologist using WHO (2005) criteria for epithelial dysplasia. At the end of the experiment, 100% of control specimens showed mild epithelial dysplasia. On laser irradiated animals, 75% of the specimens showed mild epithelial dysplasia and 25% showed moderate ones extending beyond the medium third of the epithelium. It was concluded that the use of both wavelength and a dose of 4J/cm2 may increase the severity of oral epithelial dysplasia.

Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N₂ and 5% O₂) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm² or 16 J/cm². The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm². A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm², there was a significant increase in DNA damage in irradiated cells with and without the addition of FPG. These results are indicative of the importance of both cell injury model as well as fluence when assessing the effect of phototherapy on DNA integrity.

Cryptococcosis is an infection caused by the encapsulated yeast Cryptococcus neoformans and the most afflicted sites are lung, skin and central nervous system. A range of studies had reported that photodynamic therapy (PDT) can inactivate yeast cells; however, the in vivo experimental models of cryptococcosis photoinactivation are not commonly reported. The aim of this study was to investigate the ability of methylene blue (MB) combined with a low-power red laser to inactivate Cryptococcus neoformans in in vitro and in vivo experimental models. To perform the in vitro study, suspension of Cryptococcus neoformans ATCC-90112 (106cfu/mL) was used. The light source was a laser (Photon Lase III, DMC, São Carlos, Brazil) emitting at λ660nm with output power of 90mW for 6 and 9min of irradiation, resulting fluences at 108 and 162J/cm². As photosensitizer, 100μM MB was used. For the in vivo study, 10 BALB/c mice had the left paw inoculated with C. neoformans ATCC-90112 (107cfu). Twenty-four hours after inoculation, PDT was performed using 150μM MB and 100mW red laser with fluence at 180J/cm2. PDT was efficient in vitro against C. neoformans in both parameters used: 3 log reduction with 108J/cm² and 6 log reduction with 162J/cm². In the in vivo experiment, PDT was also effective; however, its effect was less expressive than in the in vitro study (about 1 log reduction). In conclusion, PDT seems to be a helpful alternative to treat dermal cryptococcosis; however, more effective parameters must be found in in vivo studies.

In the present work we have investigated in vitro sensitivity of microorganisms P. acnes and S. epidermidis to action of red (625 nm and 405 nm) and infrared (805 nm) radiations in combination with photosensitizes Methylene Blue and Indocyanine Green.

Purpose: To investigate the effects of low level laser (LLL) irradiation for the prevention and treatment of aminoglycoside-induced vestibular ototoxicity. Materials and Methods: An organotypic culture of 2 to 4 days old rat utricular maculae hair cells was used. The cultured utricular hair cells were divided into 6 groups. Group C: the hair cells were cultured for 14 days. Group G: cultured hair cells were treated with 1 mM gentamicin (GM) for 48 hours. Group L: LLL irradiation with 670 nm diode laser 3 mW/cm² for 60 min (10.8 J/cm²)/day for 14 days. Group LG: LLL irradiation 10.8 J/ cm²/day for 2 days followed by GM insult. Group GL: treated with GM and followed by LLL irradiation 10.8 J/ cm²/day for 12 days. LGL group: LLL irradiation 10.8 J/ cm²/day for 2 days, then GM insulted, followed by the LLLT 10.8 J/ cm²/day for 10 days. The hair cells in each group were examined and counted by confocal laser scanning electron microscope on 7th and 14th days after FM1-43 staining and observed by scanning electron microscope (SEM). Results: The number of vestibular hair cells of group G was significantly less than those in group C. Group L showed no difference compared to group C. Significantly higher numbers of cells were seen in Group LG and GL comparing to group G. The cells were more in LG than group GL. Group LGL showed the most vestibular hair cells compared to the G, LG, and GL groups. SEM showed damaged hair cells in group G while they were well preserved in groups C, L, LG, GL, and LGL. Conclusion: LLL irradiation before and after GM insult on utricular hair cells were most effective to prevent and treat GM ototoxicity. This study indicates that LLL irradiation may have clinical implications to treat various vestibular and cochlear inner ear diseases.