This PDF file contains the front matter associated with SPIE Proceedings Volume 6428, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

The use of low levels of visible or near infrared light (LLLT) for reducing pain, inflammation and edema, promoting healing of wounds, deeper tissues and nerves, and preventing tissue damage by reducing cellular apoptosis has been known for almost forty years since the invention of lasers. Originally thought to be a peculiar property of laser light (soft or cold lasers), the subject has now broadened to include photobiomodulation and photobiostimulation using non-coherent light. Despite many reports of positive findings from experiments conducted in vitro, in animal models and in randomized controlled clinical trials, LLLT remains controversial. This likely is due to two main reasons; firstly the biochemical mechanisms underlying the positive effects are incompletely understood, and secondly the complexity of rationally choosing amongst a large number of illumination parameters such as wavelength, fluence, power density, pulse structure and treatment timing has led to the publication of a number of negative studies as well as many positive ones. In recent years major advances have been made in understanding the mechanisms that operate at the cellular and tissue levels during LLLT. Mitochondria are thought to be the main site for the initial effects of light and specifically cytochrome c oxidase that has absorption peaks in the red and near infrared regions of the electromagnetic spectrum matches the action spectra of LLLT effects. The discovery that cells employ nitric oxide (NO) synthesized in the mitochondria by neuronal nitric oxide synthase, to regulate respiration by competitive binding to the oxygen binding of cytochrome c oxidase, now suggests how LLLT can affect cell metabolism. If LLLT photodissociates inhibitory NO from cytochrome c oxidase, this would explain increased ATP production, modulation of reactive oxygen species, reduction and prevention of apoptosis, stimulation of angiogenesis, increase of blood flow and induction of transcription factors. In particular, signaling cascades are initiated via cyclic adenosine monophosphate (cAMP) and nuclear factor kappa B (NF-&kgr;B). These signal transduction pathways in turn lead to increased cell proliferation and migration (particularly by fibroblasts), modulation in levels of cytokines, growth factors and inflammatory mediators, and increases in anti-apoptotic proteins. The results of these biochemical and cellular changes in animals and patients include such benefits as increased healing in chronic wounds, improvements in sports injuries and carpal tunnel syndrome, pain reduction in arthritis and neuropathies, and amelioration of damage after heart attacks, stroke, nerve injury and retinal toxicity.

While Low-level Light Therapy (LLLT) has demonstrated efficacy for certain indications, some aspects of the technology are still controversial. Clinical studies on LLLT range from low quality anecdotal studies to blinded, randomized, control clinical studies. These have used a variety of wavelengths, optical powers and variations in other laser parameters. While these studies show a large range in treatment outcome, comparison of treatment efficacy between these studies with respect to light dose is all but impossible since the light dose characterization in the LLLT field has not been properly defined and is not standardized. Surface irradiance is typically used in the LLLT field as the light dose parameter, ignoring factors such as tissue optical properties, beam divergence, pulsing of the source and tissue thickness to the organ or joint of interest. Drawing on experience with light dosimetry for photodynamic and photothermal therapy, we will provide an overview of light transport and dosimetry in tissue and its implications for LLLT dosimetry. In particular, we suggest that the proper measure of dose is the light fluence rate delivered to the organ or tissue of interest, usually several millimeters below the tissue surface. We have developed a technique that provides an estimate of the subsurface fluence rate based on the diffuse reflectance measured at the tissue surface. Using Monte Carlo simulations and measurements on tissue simulating phantoms, we demonstrate that this technique can be used to predict the subsurface fluence rate to within 30% of the actual value at 3-10 mm below the tissue surface.

The effects of phototherapy on herpes lesions have been clinically demonstrated by either preventing the lesion formation or speeding their repair. The aim of this in vitro study was analyze the effect of phototherapy on epithelial cells and HSV-1 in culture. Cultures of HSV-1 and epithelial cells (Vero cell line) were used. The irradiations were done using a GaAlAs laser (660 e 780 nm, 4.0 mm²). One, two and three irradiations with 6 h-intervals were done. The experimental groups were: Control: non-irradiated; 660 nm and 3 J/cm² (2.8 sec); 660 nm and 5 J/cm² (3.8 sec); 780 nm and 3 J/cm² (1.9 sec), and 780 nm and 5 J/cm² (2.5 sec). The HSV-1 cytopatic effect and the cell viability of irradiated cultures and controls were analyzed in four different conditions: irradiation of non-infected epithelial cells; epithelial cells irradiated prior infection; virus irradiated prior infection; irradiation of HSV infected cells. The mitochondrial activity and cytopathic effects were assessed. The number of irradiations influenced the cell growth positively and proportionally, except for the 660 nm/ 3 J/cm² group. Any variation in cytopathic effects was observed amongst the experimental groups. The viability of infected cells prior irradiation was significantly higher than that of non-irradiated cultures when 2 irradiations were done. Under the experimental conditions of this study we concluded that phototherapy is capable of enhancing epithelial cell growth and prolonging cell viability of HSV-1 infected cells. Positive benefits of phototherapy could be resultant from prolongation of infected cells viability, corroborating with host defenses.

Monochromatic light therapy (MLT) is still not a clinical modality due to conflicting and low predictable outcomes. This likely is due to the mismatch between the accuracy of optical dosimetry employing the theoretically predicted or empirically found dosages and the dynamic requirements of photobiostimulation to dosage. The same doses delivered to a target area can be stimulatory or not depending on the dynamic changes of tissue optical parameters in vivo. Since the optical parameters of tissue and, hence, the radiant energy absorbed in the target area change during the treatment, it is important monitoring and coordinating the dosage with these changes. In this paper we analyze potentials of advancing to dosimetry that meets the dynamic requirements of the MLT to dosage. Both the tissue optical clearing and the feedback control of irradiation are considered. It is pointed out that the key physiological parameter influencing the variability of actual dose is the blood microcirculation. Even the optimal doses of continuous light irradiation may produce negative therapeutic effect at the time when the target tissue is depleted of blood and cannot maintain the energetic requirements of treatment. It is shown that synchronization of irradiation with the patient's pulse and breathing waves excludes cycles of negative responses and individualize the dosage.

Angiogenesis is essential for normal growth, tissue repair and regeneration. Its stimulation accelerates repair and regeneration including wound healing where these processes are delayed. Its inhibition can reduce the rate of growth of solid tumors. Phototherapy can accelerate the resolution of acute inflammation with the result that the proliferative phase of tissue repair, when angiogenesis occurs, begins earlier than in sham-irradiated controls. Evidence that angiogenesis is enhanced in dermal repair, tendon repair and bone regeneration in rodents is presented. The cellular mechanisms that control angiogenesis involve the interaction of endothelial cells, macrophages, pericytes and other cells in response, for example, to changes in the availability of oxygen in the local environment. Pericytes and macrophages modulate endothelial cell proliferation; pericytes guide endothelial cell migration. The stimulation of endothelial cell proliferation in vitro following exposure to red (660 nm) and infrared (820 nm) radiation, 15 mW, at 2-8 J/cm² is presented. 1J/cm2 was ineffective. 820 nm irradiation, 15 mW, at 8 J/cm² was observed to inhibit pericyte proliferation in vitro. Indirect effects on endothelial cell and pericyte proliferation followed stimulation of soluble mediator production by macrophages following exposure to red and infrared radiation. The potential clinical significance of the results obtained is discussed and the necessity of clinical trials emphasized.

We investigated diode laser (980 nm) evoked activation of transient receptor potential proteins (TRPV1 and TRPV2). C and A-delta (A&dgr;) nociceptor families are primarily responsible for pain mediation in the peripheral nervous system. TRPV1 proteins have been associated with heat evoked pain in C fibers while A&dgr; fibers have been associated with TRPV2. Diode laser stimulation allows a margin of safety between non-invasive activation and damage ^{19, 22, 34}. Laser pulses (20-50 ms, 0.1-10 W, 980 nm) were used to stimulate: A) in vitro: excised patches from HEK293 cells expressing TRPV1; B) in vitro: rat DRG nociceptors expressing either TRPV1 or TRPV2; and C) in vivo: C-fibers of the rat saphenous nerve (SN) trunk. Cell currents were recorded using standard patch clamp methods. The SN was also stimulated electrically with bipolar electrodes. Stimulation (20-50 ms) of HEK and DRG cells expressing TRPV1 was highly reproducible. Activation and peak currents were achieved at estimated peak temperatures of 55°C and 70°C. Threshold activation was also observed in DRG neurons expressing TRPV2. The conduction velocity for laser-activated saphenous nerve afferents was in the C fiber range (0.5-1 m/s). Electrically stimulated nerve contained stimulation artifacts and complex neural components with conduction velocities ranging from 0.3-30 m/s. Diode laser activation of TRPV1 protein is a reproducible and effective means to probe TRP activity in both in vivo and in vitro preparations

In this study, a preliminary approach for pain relief using a novel pulsated LED device was conducted. 12 patients were treated with a Photopuncture device designed by SORISA, which consisted in a 10-channel LED system at 617 nm. 15 patients with different pain localizations were treated: cervicobrachialgia (3 cases), lumbago / sciatica (4 cases), gonalgia (3 cases), cephalalgia (2 cases), talalgia (1 case), epicondylitis (1 case) and trigeminal neuralgia (1 case). To characterize the pain level, the Categorical Pain Scale (none (0), mild (1-3), moderate (4-6) and severe (7-10)) was used. Just patients with severe pain (7-10) were treated. Patients were treated twice a week for 25 minutes; 5 to 8 sessions were given at the following treatment parameters: 10 mW per channel pulsed at 60 Hz with a 50 % duty cycle. The total dose for point was 7.5 J. To characterize the response to the treatment, the results were classified as: "no result", no changes in pain degree; "poor", pain decreased one category; "good", pain decreased two categories; "very good", complete healing (no pain). The results were: 1 case with "very good" result; 11 cases with "good" result; 3 cases with "poor" result; and 0 cases with "no result". We conclude that the Photopuncture led device may be a good alternative to classical Acupuncture in pain relief, although further experimentation is required.

This study investigates whether low level light treatment (LLLT) can enhance the expression of Peripheral-type mitochondrial benzodiazepine receptors (PBRs) on the glioma-derived tumour cell line, CNS-1, and by doing so promote the synthesis of protoporphyrin IX (PpIX) and increase the photodynamic therapy (PDT)-induced cell kill using 5-aminolevulinic acid (ALA). The endogenous photosensitizer, (PpIX) and related metabolites including coproporphyrin III are known to traffic via the PBRs on the outer mitochondrial membrane on their passage into or out of the mitochondria. Astrocyte-derived cells within the brain express PBRs, while neurons express the central-type of benzodiazepine receptor. CNS-1 cells were exposed to a range of differing low-level light protocols immediately prior to PDT. LLLT involved using broad-spectrum light or monochromatic laser light specific to 635 or 905 nm wavelength. Cells (5&mgr;10⁵) were exposed to a range of LLLT doses (0, 1 or 5 J/cm²) using a fixed intensity of 10 mW/cm² and subsequently harvested for cell viability, immunofluorescence or western blot analysis of PBR expression. The amount of PpIX within the cells was determined using chemical extraction techniques. Results confirm the induction of PBR following LLLT is dependent on the dose and wavelength of light used. Broadspectrum light provided the greatest cell kill following PDT, although LLLT with 635 nm or 905 nm also increased cell kill as compared to PDT alone. All LLLT regimens increased PBR expression compared to controls with corresponding increases in PpIX production. These data suggest that by selectively increasing PBR expression in tumour cells, LLLT may facilitate enhanced cell kill using ALA-PDT without damaging surrounding normal brain.

Over the past few decades, many efforts were devoted to study low power laser and cellular interaction. Some of the investigations were performed on cell populations. In this work fiber-optic based nano-probe is used for the precise delivery of laser light on to a single cell and the mechanism of light interaction with the cell during irradiation was studied. A human skin fibroblast cell line was utilized in this investigation. The human fibroblasts were irradiated under two different schemes of exposure: (1) entire cell population was irradiated within a Petri dish using a fan beam, (2) laser energy was precisely delivered on to a single cell using fiber-optic nano-probe. Studies were conducted by variation of laser intensity, exposure time, and the energy dose of exposure. Proliferative effect of laser irradiation was determined through cell counting for both exposure schemes. Enhancement of the rate of proliferation was observed to be dependent on laser parameters and method of laser delivery. Variation of total energy dose had greater effect on the enhancement of the rate of cellular proliferation compared to that of laser intensity. The photobiostimulative effect was also observed to have a finite life-time. Fluorescent life-time imaging of reactive oxygen species (ROS) was performed during the single cell exposure method. ROS generation was found to depend strongly on both laser energy doses and irradiation time. It is demonstrated in this communication that by using specially engineered nano-probes, laser light can be precisely delivered on to a targeted single cell.

The role of low light intensity in suppressing metabolic activity of transformed cell lines was investigated through the applications of a 1,552nm wavelength pulsed picosecond laser. Human malignant glioblastoma, human leukemia HL-60, and the NIH 3T3 cell lines were used. The cells were grown in 96 well plates and exposed in their respective growth culture media with 10% (v/v) fetal bovine serum under various fluence exposure conditions ranging from 0.115 - 100 J/cm². All cell lines were exposed at a constant average intensity value of 0.115 W/cm²; 25 kHz repetition rate with 1.6 micro-joule per pulse; pulse duration = 2.93 picosecond. The human malignant glioblastoma and the HL-60 cell lines exhibited a monotonic decline in metabolic activity (down 50 - 60%) relative to their respective sham exposed control counterparts between the fluence values of 0.115 J/cm² to 10 J/cm². The NIH 3T3 cells exhibited a maximum suppression of metabolic activity at the fluence value of 50 J/cm². Metabolic activity was measured through the colorimetric MTS metabolic assay. Interestingly, for all cell lines the metabolic activity was found to return back to the sham exposed control levels as the fluence of exposure was increased up to 100J/cm².

It has been known for many years that low level laser (or light) therapy (LLLT) can ameliorate the pain, swelling and inflammation associated with various forms of arthritis. Light is absorbed by mitochondrial chromophores leading to an increase in ATP, reactive oxygen species and/or cyclic AMP production and consequent gene transcription via activation of transcription factors. However, despite many reports about the positive effects of LLLT in medicine, its use remains controversial. Our laboratory has developed animal models designed to objectively quantify response to LLLT and compare different light delivery regimens. In the arthritis model we inject zymosan into rat knee joints to induce inflammatory arthritis. We have compared illumination regimens consisting of a high and low fluence (3 J/cm² and 30 J/cm²), delivered at a high and low irradiance (5 mW/cm2 and 50 mW/cm²) using 810-nm laser light daily for 5 days, with the effect of conventional corticosteroid (dexamethasone) therapy. Results indicated that illumination with 810-nm laser is highly effective (almost as good as dexamethasone) at reducing swelling and that longer illumination time was more important in determining effectiveness than either total fluence delivered or irradiance. Experiments carried out using 810-nm LLLT on excisional wound healing in mice also confirmed the importance of longer illumination times. These data will be of value in designing clinical trials of LLLT.

The aim of this study was to analyze the effects of phototherapy with low intensity laser on the inflammatory reaction after rat brain injury. Cryogenic injury was performed at the brain of 16 male Wistar rats (250-300g) using a cooper probe at -80º C. Immediately, 24 h and 48 h later, the rats received laser irradiation using a GaAlAs laser (830 nm, 100 mW). The samples were randomly divided into four groups (n= 4 per group): A: control (non- irradiated); B: energy density of 14.28 J/cm²; C: 28.57 J/cm²; D: 42.85 J/cm². Three days later, the cerebral vascular permeability and the inflammatory cells at the trauma site were evaluated. For vascular permeability analysis, 2 h prior sacrifice an intra vascular injection of Evans blue stain was done in the rats. For inflammatory cells counting, frozen samples were sectioned and the histological slides were stained with Giemsa. The data were compared by either ANOVA or Kruskall-Wallis complemented by the Dunn's test. The irradiated groups presented higher cerebral vascular permeability than controls (A: 2.6 ± 0.8; B:12.0 ± 2.0; C: 13.1 ± 4.1, and D: 12.4 ± 1.8; p=0.016). The inflammatory cell numbers of irradiated samples were similar to controls (A: 65 ± 6; B:85 ± 9; C: 84 ±14, and D: 83 ± 3; p=0.443). The data showed that phototherapy with low intensity laser modulates the inflammatory reaction in the brain by increasing the cerebral vascular permeability after a cryogenic trauma.

This is a clinical presentation demonstrating the efficacy of laser therapy in the treatment of patients presenting with trauma to both the hard and soft tissue in the orofacial region. The use of laser therapy aids the management of these cases where the patients often present with anxiety and a low pain threshold. The outcomes in these cases indicate good patient acceptance of the treatment, enhanced repair and tissue response suggesting that this form of treatment can be indicated for these patients. A combination of hard and soft lasers are used for the comprehensive dental management and treatment of these cases. The lasers used are a 810nm diode and an Er.CrYSGG.

Low levels of light have been reported to change human physiologic activities. In our research we focused at an extreme situation and utilized human's own ultra-weak light for radiation. Human photon emission was studied after dark-adaptation utilizing a sensitive, cooled, moveable photomultiplier system. Data showed that palm and dorsum of the hands showed most emission. Human light was reflected by a red color filter in close proximity but not touching the body of the dark-adapted subject. Photon emission from the palm was recorded before and after the filter was placed at 3 cm from the skin between the palm and the photomultiplier. After removing the filter the photomultiplier recorded increased emission compared to emission prior to exposure. The effect faded away in 7 min. To study whether this effect is only local, photon emission of the hand dorsum was recorded and the filter was placed 3 cm below the palm. Such exposure also resulted in increased emission that faded away. In this protocol, photon emission of the dorsum was also recorded during the period that the palm was exposed to the filter. Photon emission increased immediately after positioning the filter. As a first step to an explanation we discuss recent studies on the optical properties of human photon emission. The photon signal has non-classical features and is well described by the photon signal in a coherent state. It is hypothesized that the human photon field carries information on the chemical reactions in the physiologic state.

The advantages of the modified Nakatani method for diagnostics of energetic condition of meridians are described. Also the perspectives of its clinical application for dynamic patient monitoring in treatment process are considered. The model of a biological feedback through measurement of electric conductivity of a skin at pulse current is represented with its mathematical and software base. The results of clinical tests of the treatment-and-diagnostic complex (TDC) functioning in correspondence with the modified Nakatani method are analyzed.