Photobiomodulation therapy can change actin filaments of 3T3 mouse fibroblast.

The purpose of this study was to investigate the effects that photobiomodulation therapy might produce in cells, in particular, related to their structure. Thus, this paper presents the results of morphological changes in fibroblasts following low-intensity light illumination. Mouse fibroblasts were grown on glass coverslips on either 4 kPa or 16 kPa gels, to mimic normal tissue conditions. Cells were photo-irradiated with laser light at either 625 nm or 808 nm (total energies ranging from 34 to 47 J). Cells were fixed at 5 min, 1 h, or 24 h after photo-irradiation, stained for both actin filaments and the cell nucleus, and imaged by confocal microscopy. A non-light exposed group was also imaged. A detailed analysis of the images demonstrated that the total polymerized actin and number of actin filaments decrease, while the nucleus area increases in treated cells shortly after photo-irradiation, regardless of substrate and wavelength. This experiment indicated that photobiomodulation therapy could change the morphological properties of cells and affect their cytoskeleton. Further investigations are required to determine the specific mechanisms involved and how this phenomenon is related to the photobiomodulation therapy mechanisms of action.

Skin color and tissue thickness effects on transmittance, reflectance, and skin temperature when using 635 and 808 nm lasers in low intensity therapeutics.

OBJECTIVE: To examine the role of skin color and tissue thickness on transmittance, reflectance, and skin heating using red and infrared laser light. METHODS: Forty volunteers were measured for skin color and skin-fold thickness at a standardized site near the elbow. Transmittance, reflectance and skin temperature were recorded for energy doses of 2, 6, 9, and 12 Joules using 635 nm (36 mW) and 808 nm (40 mW) wavelength laser diodes with irradiances within American National Standards Institute safety guidelines (4.88 mm diameter, 0.192 W/cm2 and 4.88 mm diameter, 0.214 W/cm2 , respectively). RESULTS: The key factors affecting reflectance to an important degree were skin color and wavelength. However, the skin color effects were different for the two wavelengths: reflectance decreased for darker skin with a greater decrease for red light than near infrared light. Transmittance was greater using 808 nm compared with 635 nm. However, the effect was partly lost when the skin was dark rather than light, and was increasingly lost as tissue thickness increased. Dose had an increasing effect on temperature (0.7-1.6°C across the 6, 9, and 12 J doses); any effects of wavelength, skin color, and tissue thickness were insignificant compared to dose effects. Subjects themselves were not aware of the increased skin temperature. Transmittance and reflectance changes as a function of energy were very small and likely of no clinical significance. Absorption did not change with higher energy doses and increasing temperature. CONCLUSION: Skin color and skin thickness affect transmittance and reflectance of laser light and must be accounted for when selecting energy dose to ensure therapeutic effectiveness at the target tissue. Skin heating appears not to be a concern when using 635 and 808 nm lasers at energy doses of up to 12 J and irradiance within American National Standards Institute standards. Photobiomodulation therapy should never exceed the American National Standards Institute recommendation for the maximum permissible exposure to the skin. Lasers Surg. Med. 50:291-301, 2018. © 2017 Wiley Periodicals, Inc.

Low-intensity laser therapy efficacy evaluation in mice subjected to acute arthritis condition.

Acute arthritis is an inflammation that affects many joints. The principal treatment options comprise drugs (corticosteroids), invasive and painful surgery. The objective of this study was to evaluate the efficacy of low intensity laser therapy (LILT), a non-invasive treatment, in a murine model of acute inflammation model. 48 mice received a synovial injection of Zymosan A into one knee. Mice were treated with LILT by three different wavelengths, either as a single (S) or dual (D) application immediately after the injury or after 24h following initiation of an inflammatory response. The histological analysis aimed at identifying inflammatory infiltrate and the structure of the articular surfaces as an indicator of a long-term damage due to inflammation. Statistical analysis (Kruskal-Wallis test), did not allow to reject the null hypothesis. However, LILT promoted histological alterations in some treatment groups. Histological evidence (Median and confidence interval) of anti-inflammatory effects was especially noticeable in knees of mice irradiated with lasers emitting moderate intensity and continuous 660nm (S=18.5 (11.4; 27.6); D=16.0 (6.93; 27.0)) and high intensity and pulsed 905nm (S=17.5 (10.2; 24.79), with decrease of the resorbed region. However, the 905nm pulsed laser was responsible for exacerbation of inflammation for multiple LILT sessions with a short delay (D=45.0 (22.84; 63.83)), tending to aggravate the resorption of the articular surface (p<0.05). LILT showed signs of an anti-inflammatory effect when applied once, but promoted increased resorptive area when used for two sessions, indicating the importance of a controlled LILT protocol to reach therapeutic effects.

Low-intensity laser therapy efficacy evaluation in FVB mice subjected to acute and chronic arthritis.

Rheumatoid arthritis, an autoimmune inflammation, has a high prevalence in the population, and while therapy is available, it required often injection of drugs causing discomfort to patients. This study evaluates the clinical and histological effect of low-intensity laser therapy (LILT) as an alternative treatment, in a murine model of acute and chronic inflammation. FVB mice received either a Zymosan A injection into one knee joint inducing acute inflammation, followed after 15 min or 24 h by LILT or a collagen bovine type II injection emulsified in "Freund's Complete Adjuvant" to induce chronic arthritis, followed at 4 weeks with multiple LILT sessions. LILT mediated by either 660, 808, or 905 nm and tissue response was evaluated based on clinical symptoms and histological analysis of inflammatory infiltrate and damage to the articular surfaces. LILT can be effective in elevating clinical symptoms, so Kruskal-Wallis testing indicated no significant differences between knees affected by acute arthritis and treated once with LILT and an injured knee without treatment (p > 0.05) for 660 and 808 nm with some improvements for the 905-nm LILT. Mice receiving two treatments for acute arthritis showed exacerbation of inflammation and articular resorption following therapy with a 660-nm continuous laser (p < 0.05). For chronic inflammation, differences were not noted between LILT treated and untreated injured knee joints (p > 0.05). Among the lasers, the 905 nm tends to show better results for anti-inflammatory effect in acute arthritis, and the 660 nm showed better results in chronic arthritis. In conclusion, LILT wavelength selection depends on the arthritis condition and can demonstrate anti-inflammatory effects for chronic arthritis and reduced resorption area in this murine model.

Effects of low intensity laser irradiation during healing of infected skin wounds in the rat.

BACKGROUND AND OBJECTIVE: Low intensity laser irradiation remains a controversial treatment for non-healing wounds. This study examines the effect of low intensity light on healing of infected skin wounds in the rat. MATERIALS AND METHODS: Wounds on the rat dorsum were inoculated with Pseudomonas aeruginosa. Wounds were irradiated or sham-irradiated three times weekly from day 1 to 19 using 635-nm or 808-nm diode lasers delivering continuous wave (CW) or intensity modulated (3800 Hz) laser radiation, all at radiant exposures of 1 and 20 J/cm2. Wound area and bacterial growth on the wound surface were evaluated three times a week. Histological and immunohistochemical analyses were performed at day 8 and 19. RESULTS: Wounds that were irradiated using a wavelength of 635 nm (1 and 20 J/cm2) or intensity modulated 808-nm laser light at 20 J/cm2 were smaller in area at day 19 than the sham-irradiated controls (achieved significance level=0.0105-0.0208) and were similar to controls in respect of bacterial growth. The remaining light protocols had no effect on wound area at day 19 although they increased Staphylococcus aureus growth across the time line compared with controls (p<0.0001 to p<0.004). CW 808-nm light at 20 J/cm2 significantly delayed half-heal time. Histological and immunohistochemical analyses supported wound closure findings: improved healing was associated with faster resolution of inflammation during the acute phase and increased signs of late repair at day 19. Significant inflammation was seen at day 19 in all irradiated groups regardless of radiant exposure, except when using 635 nm at 1 J/cm2. CONCLUSIONS: Red light improved healing of wounds. Only one 808-nm light protocol enhanced healing; lack of benefit using the remaining 808-nm light protocols may have been due to stimulatory effects of the light on S. aureus growth.

Effects of low intensity laser irradiation during healing of skin lesions in the rat.

OBJECTIVE: To determine whether laser light can improve healing of skin wounds by killing wound bacteria while simultaneously accelerating host tissue activity. MATERIALS AND METHODS: Wounds on the rat dorsum were irradiated or sham-irradiated three times weekly from days 1 to 19 using 635 or 808 nm diode lasers at 1 or 20 J/cm(2). Wound area and bacterial growth were evaluated three times weekly. Histological analysis was performed on days 8 and 19. Immunohistochemical analysis was performed on day 19. RESULTS: Wounds that were irradiated using 635 nm light at 1 J/cm(2) healed similarly to controls. Wounds that were irradiated using 808 nm (1 and 20 J/cm(2), P

Three-dimensional fluence rate measurement and data acquisition system for minimally invasive light therapies.

Light based therapies such as photodynamic therapy are in need of advanced tools for light fluence rate dosimetry and monitoring within the context of therapy planning and light delivery to ensure maximum treatment efficacy. The use of a single, multisensor fiber-based fluorescent probe capable of performing spatially resolved fluence rate measurements along an axis was demonstrated. This work extends the previous technique and describes a fluence rate quantification system able to employ up to 12 multisensor probes to simultaneously measure fluence rate distribution throughout a 3D treatment volume. The system optoelectronics provides for sensor calibration, data acquisition, and weighted least-squares processing to extract localized fluence rate information in real-time. Core components include an integrating cylinder for source sensor calibration, a 2D back thin CCD detector for sensor signal detection from multiple probes, high-speed data acquisition card, and custom software for real-time extraction of fluence rate information from all sensors.

In vivo effects of low level laser therapy on inducible nitric oxide synthase.

BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) has been demonstrated to modulate inflammatory processes with evidence suggesting that treatment protocol, such as wavelength, total energy, and number of treatments determine the clinical efficacy. In this study, the effects of LLLT mediated by different wavelengths and continuous versus pulsed delivery mode were quantified in a transgenic murine model with the luciferase gene under control of the inducible nitric oxide synthase (iNOS) expression. STUDY DESIGN/MATERIALS AND METHODS: LLLT modulated iNOS gene expressed in the acute Zymosan-induced inflammation model is quantified using transgenic mice (FVB/N-Tg(iNOS-luc)). Here an energy density of 5 J cm(-2) at either 635, 660, 690, and 905 nm in continuous wave mode and at 905 nm for short pulse delivery were evaluated. Age of the animals was determined as additional modulating the inflammatory response and the LLLT efficacy for some treatment protocols. RESULTS: Animals younger than 15 weeks showed mostly reduction of iNOS expression, while older animals showed increased iNOS expression for some LLLT protocols. Intensity and time course of inducible nitric oxide expression was found to not only depend on wavelength, but also on the mode of delivery, continuous, or pulsed irradiation. CONCLUSION: LLLT exhibit different effects in induced inflammatory process according to different wavelengths and wave mode. Upregulation of iNOS gene following 905 nm pulsed wave suggests a different mechanism in activating the inflammatory pathway response when compared to the continuous wave.

In vivo study of the inflammatory modulating effects of low-level laser therapy on iNOS expression using bioluminescence imaging.

This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (lambda = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm(-2), 200 s, 5 J cm(-2)) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.

Performance evaluation of cylindrical fiber optic light diffusers for biomedical applications.

BACKGROUND AND OBJECTIVES: Dosimetry for intracavity and interstitial light delivery requires next to the knowledge of tissue optical properties and models describing light propagation in tissue also exact knowledge of the spatial light source emission characteristics. However, the emission characteristics of cylindrical diffusers are often ill defined by the manufacturer, and not regularly determined by the end user, thus limiting the attainable dosimetry accuracy. STUDY DESIGN/MATERIALS AND METHODS: Commercial cylindrical diffusers, with active diffusive lengths of 1-2 cm and outer diameters of

Effects of low-level laser therapy (LLLT) of 810 nm upon in vitro growth of bacteria: relevance of irradiance and radiant exposure.

OBJECTIVE: The aim of this study was to investigate the irradiance-dependency of low-level laser therapy (LLLT) effects on bacterial growth. BACKGROUND: LLLT is applied to open wounds to improve healing; however, its effect on wound bacteria is not well understood. MATERIALS AND METHODS: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were irradiated using a wavelength of 810 nm at irradiances of 0.015 W/cm2 (0-50 J/cm2) and 0.03 W/cm2 (0-80 J/cm2). Bacteria were counted after 20 h of incubation. RESULTS: LLLT effects varied significantly with species. P.aeruginosa growth decreased overall dependent on an interaction of irradiance and radiant exposure; greatest inhibition was produced using high irradiance delivering radiant exposures in the range of 1-20 J/cm2 (p = 0.001-0.04). In contrast, E. coli growth increased overall (p = 0.01), regardless of irradiance; greatest effects were produced using low radiant exposures (1-20 J/cm2). There was a main effect for irradiance (p = 0.03) on S. aureus growth; however, growth was not different compared with controls. Additional analysis showed that there were differences in growth of P.aeruginosa when comparing samples that were matched by exposure times (66, 329, 658, 1316, 1974, and 2632 sec) rather than radiant exposure; this suggests that irradiance rather than exposure time was the significant factor in P. aeruginosa inhibition. CONCLUSION: These findings have immediate relevancy in the use of LLLT for infected wounds. Exposure to 810-nm irradiation (0.03 W/cm2) could potentially benefit wounds infected with P. aeruginosa. However, increased E. coli growth could further delay recovery.

Effects of 810 nm laser irradiation on in vitro growth of bacteria: comparison of continuous wave and frequency modulated light.

BACKGROUND AND OBJECTIVES: Low intensity laser therapy may modify growth of wound bacteria, which could affect wound healing. This study compares the effects on bacteria of 810 nm laser using various delivery modes (continuous wave or frequency modulated light at 26, 292, 1000, or 3800 Hz). STUDY DESIGN/MATERIALS AND METHODS: Staphylococcus (S.) aureus, Escherichia (E.) coli, and Pseudomonas (P.) aeruginosa were plated on agar and then irradiated (0.015 W/cm(2); 1-50 J/cm(2)) or used as controls (sham irradiated); growth was examined after 20 hours of incubation post exposure. RESULTS: There were interactions of species and modulation frequency in the overall effects of irradiation (P = 0.0001), and in the radiant exposure mediated effects (P = 0.0001); thus individual frequencies and each bacterium were analysed separately. Bacteria increased following 3800 Hz (P = 0.0001) and 1000 Hz (P = 0.0001) pulsed irradiation; at particular radiant exposures P. aeruginosa proliferated significantly more than other bacteria. Pulsed laser at 292 and 26 Hz also produced species-dependent effects (P = 0.0001; P = 0.0005); however, the effects for different radiant exposures were not significant. Bacterial growth increased overall, independent of species, using continuous mode laser, significantly so at 1 J/cm(2) (P = 0.02). Analysis of individual species demonstrated that laser-mediated growth of S. aureus and E. coli was dependent on pulse frequency; for S. aureus, however, there was no effect for different radiant exposures. Further tests to examine the radiant exposure effects on E. coli showed that growth increased at a frequency of 1000 Hz (2 J/cm(2); P = 0.03). P. aeruginosa growth increased up to 192% using pulsed irradiation at 1000-3800 Hz; whereas 26-292 Hz laser produced only a growth trend. CONCLUSIONS: The findings of this study point to the need for wound cultures prior to laser irradiation of infected wounds. Similar investigations using other common therapeutic wavelengths are recommended.

Effects of 630-, 660-, 810-, and 905-nm laser irradiation delivering radiant exposure of 1-50 J/cm2 on three species of bacteria in vitro.

OBJECTIVE: To examine the effects of low-intensity laser therapy (LILT) on bacterial growth in vitro. BACKGROUND DATA: LILT is undergoing investigation as a treatment for accelerating healing of open wounds. The potential of coincident effects on wound bacteria has received little attention. Increased bacterial proliferation could further delay recovery; conversely inhibition could be beneficial. MATERIALS AND METHODS: Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus were plated on agar and then irradiated with wavelengths of 630, 660, 810, and 905 nm (0.015 W/cm(2)) and radiant exposures of 1-50 J/cm(2). In addition, E. coli was irradiated with 810 nm at an irradiance of 0.03 W/cm(2) (1-50 J/cm(2)). Cells were counted after 20 h of incubation post LILT. Repeated measures ANOVA and Tukey adjusted post hoc tests were used for analysis. RESULTS: There were interactions between wavelength and species (p = 0.0001) and between wavelength and radiant exposure (p = 0.007) in the overall effects on bacterial growth; therefore, individual wavelengths were analyzed. Over all types of bacteria, there were overall growth effects using 810- and 630-nm lasers, with species differences at 630 nm. Effects occurred at low radiant exposures (1-20 J/cm(2)). Overall effects were marginal using 660 nm and negative at 905 nm. Inhibition of P. aeruginosa followed irradiation using 810 nm at 5 J/cm(2) (-23%; p = 0.02). Irradiation using 630 nm at 1 J/cm(2) inhibited P. aeruginosa and E. coli (-27%). Irradiation using 810 nm (0.015 W/cm(2)) increased E. coli growth, but with increased irradiance (0.03 W/cm(2)) the growth was significant (p = 0.04), reaching 30% at 20 J/cm(2) (p = 0.01). S. aureus growth increased 27% following 905-nm irradiation at 50 J/cm(2). CONCLUSION: LILT applied to wounds, delivering commonly used wavelengths and radiant exposures in the range of 1-20 J/cm(2), could produce changes in bacterial growth of considerable importance for wound healing. A wavelength of 630 nm appeared to be most commonly associated with bacterial inhibition. The findings of this study might be useful as a basis for selecting LILT for infected wounds.