1. Blue LED Treatment of Superficial Abrasions

Intro: This study utilizes blue LEDs in the 400-450 nm range to irradiate bleeding wounds on rats. Photocoagulation results after hemoglobin is exposed to light and heat.

Methods: One high-power LED in the blue region, total radiative power output of 500 mW, 1.0 W/cm2. n=10. four wounds on each rat. two as control. specimens of the LED treated and untreated dorsal rat skin were harvested from the dead rats and cryo-sectioned.

Results: No adverse effects were found and microscopy shows that complete restoration occurred by 8 days. Untreated wounds had increased  fibroblast and myofibroblasts (still healing).

Conclusion: Possible solution for coagulation in superficial abrasions. Localized thermal effect. Shorter healing times.

2. Antimicrobial Blue Light Therapy for Multidrug-Resistant Acinetobacter baumannii Infection in a Mouse Burn Model: Implications for Prophylaxis and Treatment of Combat-related Wound Infections

Intro: The need for treatments that do not induce resistant strains is high. Blue light is possible treatment method. This study tests blue light therapy on multidrug resistant A. Baumannii in mouse burns.

Methods: A mouse model of third degree burn infected with A. Baumannii. Blue light at 415 nm. A single exposure of blue light was initiated 30 minutes after bacterial inoculation to inactivate. total illumination duration, 62 minutes. 19.5 mW/cm2 for cell culture experiments and to 14.6 mW/cm2 for in vivo experiments.

Results: A. Baumannii experienced >4log(10) inactivation after single exposure to 70.2J/cm2 while keratinocytes had 0.1 log(10) viability loss.  TEM shows ultrastructural damage to the cells after single exposure of 86.4 J/cm2. Fluorescence spectrum suggests that porphyrins within the bacteria are responsible for the antimicrobial effects. Bacterial bioluminescence of treated mice was completely eliminated after single exposure to 55.8J/cm2 (4.4 log(10) reduction for treated 0.14 log(10) for untreated mice). There was a 16.5-fold reduction of the AUC (bacteria burden) for treated mice. There was a tendency for increased bacterial inactivation with number of cycles therefore no resistance developed. At 195 J/cm2 resulted in no apoptotic cells in the epidermis immediately after exposure.

Conclusion: Can use blue light to inactivate bacteria. No evidence of bacteria developing a resistance to blue light. Useful for multi-drug resistant wound infections.

3. Blue Light Eliminates Community-Acquired Methicillin-Resistant Staphylococcus aureus in Infected Mouse Skin Abrasions

Intro: MRSA is of big concern due to virulence and resistant characteristics. New treatment methods are needed to control MRSA infections. This study tests the effects of blue light at 415 nm on CA-MRSA in vivo.

Methods: 415 nm. 19.5 mW/cm2 in vitro, 15.0 mW/cm2 in vivo. Total blue light exposure of up to 108 J/cm2 (120 min illumination).

Results: Significant reduction in bacteria in vitro occurred at 56.1 J/cm2 . At an exposure of  112.2 J/cm2 there was  4.75log(10) bacterial inactivation. Keratinocytes have a 25-fold slower inactivation rate than the MRSA. TEM shows disruption in the cytoplasm of the treated bacteria cells. Mouse abrasions infected with 3×106 CFU were rid of bacterial luminescence after 41.4 J/cm2  (15 mW/cm2 for 46min) when treated 30min after inoculation. It took 108 J/cm2 to eliminate luminescence when the treatment started 24 hours after inoculation. Later stage infections are more resistant to therapy. Bacterial regrowth occurred in both groups after 24 hours.

Conclusion: in vitro studies show that USA 300 LAC was much more susceptible to blue light inactivation than were HaCaT cells. bacterial inactivation in vivo (in mice) using blue light was more efficient than that using bacterial suspensions in vitro. infections treated on day 1 were more resistant to blue light therapy than they were on day 0. On day 1 after bacterial inoculation, the infections were fully established, and USA300 LAC existed predominantly as biofilms. The biofilm matrix could block blue light and render bacterial cells less susceptible to blue light.

4. Effect of NASA light-emitting diode (LED) irradiation on wound healing

Intro: LEDs developed by NASA stimulate processes in the mitochondria. Their previous studies show that wavelengths of 680 nm, 730 nm, and 880nm were most effective. Their aim is to test the validity of LED therapy to treat wounds.

Methods: 680 nm, 730 nm, and 880 nm. fibroblasts and osteoblasts, 24 well plates with a well diameter of 2 square centimeters. L-6 musculoskeletal cell line (rat derived), LED light at both combined wavelengths and individual wavelengths (670nm, 728 nm, and 880nm), energy densities of 4 and 8J/cm2, and an intensity of 50mW/cm2. Normal human epithelial cell line, 670 nm and 4J/cm2 energy density (50mW/cm2 power density), wavelength of 880nm and 8J/cm2 energy density (53 mW/cm2 power density). HaCAT epithelial cells were seeded in two 12-well tissue culture plates with 600?l DMEM containing 10% FBS, 1% penicillin/streptomycin, and 6?l L-proline [2,3-3H]. The media was free of non-essential amino acids. One plate was used as the control and the other was treated with the 670 nm NASA-LED at 8J/cm2. Measured collagen synthesis of the HaCAT epithelial cells. Model of an ischemic wound in rats, effects of NASA LED technology and hyperbaric oxygen therapy (HBO), the control (no LED or HBO), HBO only, LED (880 nm) only, and LED and HBO in combination. Wound healing impaired type 2 diabetic mouse model, type 2 diabetic mice with excisional skin wounds were treated with LEDs at individual wavelengths of 680 nm, 730 nm, and 880 nm at 4J/cm2 and 50mW/cm2. An LED array with 3 wavelengths combined in a single unit (670 nm, 720 nm, and 880 nm) was delivered to Naval Special Warfare Group-2 (SEALS) in Norfolk, VA. Treatment was with 4J/cm2.

Results: In vitro, LED irradiation accelerated growth of fibroblasts and osteoblasts for 2-3 days then cells reached full confluence and growth plateaued. In L-6 musculoskeletal rat cells, 8J/cm2 at 50mW/cm2 increased cell proliferation by 140% over the control. In human epithelial cells, 880nm at 8J/cm2  resulted in a 171% increase in growth (53 mW/cm2 power density). HaCAT epithelial cells were also shown to synthesize more than twice the collagen of control cells using 670 nm light at 8J/cm2 (50 mW/cm^2). In rats, wounds treated with 880nm and HBO were 36% smaller than the control by day 7. LEDs alone resulted in 20% smaller wounds. The gap between the treatment groups gets smaller because wound healing slows down near completion. In humans, there was a 50% increase in laceration healing compared to the control when using 670 nm, 720 nm, and 880 nm combined.

Conclusion: LED therapy is a viable treatment option.

5. Light therapy by blue LED improves wound healing in an excision model in rats

Intro: Blue light has shown to treat wounds in pig models and small animal models. LEDs have been presented as a comfortable, potentially highly selective light source for therapy. This study aims to test the effects of 630 nm and 470 nm light on in vivo wound healing in rats.

Methods: Circular excision wounds were surgically created on the dorsum of each rat. Excisions on either the left or right side were illuminated post-OP and on five consecutive days for 10 min by LED at 470 nm or 630 nm with an intensity of 50 mW/cm2. On day 7 post-OP, planimetric and histological parameters were analysed, as well as expression of keratin-1, keratin-10 and keratin-17 on mRNA level.

Results: Wound sizes were calculated at day 3 and 7 post-OP. There were no significant differences at day 3 however, on day 7 wound size decreased by 50% with blue light. However, red light seemed to delay wound closure (not very significant). Granulation was not affected however epithelialization was increased. Red light decreased Krt-1 mRNA expression at day 7 compared to no light exposure (little effect with blue light). Krt-10 mRNA levels were increased in both groups with a significant difference in the red group. Krt-17 was also significantly increased in the red light group compared to no light exposure.

Conclusion: Blue light significantly influences wound healing. Furthermore, our data suggest that light therapy can play an important role in normotrophic wound healing by affecting keratin expression. Illumination would provide an easily applicable, safe and cost-effective treatment of surface wounds.

6. Effect of NASA Light-Emitting Diode Irradiation on Molecular Changes for Wound Healing in Diabetic Mice

Intro: LED therapy has been shown to work in mice and small animal models. LEDs with wavelengths of 630-880nm have been shown to penetrate into the skin and can be used to treat wounds. This study aims to investigate the molecular changes that occur in a diabetic mouse model after irradiation to LED light.

Methods: Eighty genetically diabetic mice split evenly into two groups. One group control. Other group treated with 670nm LED given daily at a fluence of 4 J/cm2 for 14 days. Once per day, 670nm LED given at a power of 28 mW/cm2 for 2 min and 24 sec to achieve a dose of 4 J/cm2. Each of the two groups was subdivided into four groups of 10 animals each. On days 2, 4, 7, and 14 animals were sacrificed and samples were collected for cDNA microarray analysis.

Results: Mice treated with LEDs had increased healing rates compared to surgical controls. In addition, basement membrane and tissue regeneration genes were significantly upregulated in the LED group. These include collagen IV, laminin, nidogen, myosin, and other proteoglycans. Galectin-7, which is responsible for stratification of the epthelia was upregulated at day 2 and continued to increase after 14 days of treatment. Fibroblast growth factors 7 and 12 were also upregulated by day 2. Genes for TGF-beta and thrombospondin 1 (TSP-1) were upregulated later by day 14. Receptors for cytokines such as interleukin-1 , IL-10, MIP-2, and proapoptosis associated genes were downregulated.

Conclusion: Biochemical mechanism by which LED enhances the wound healing process is not known. Current theory is that infrared light is absorbed by some photoreceptors which then triggers a cascade of reactions in a cell. The major biological photoaccpetors in the near-infrared range have been determined to be hemoglobin, myoglobin, and cytochrome oxidase. LED treatment effectively energized the cells by stimulating their cytochrome oxidase and triggered a cascade of cellular and molecular events that have significant biological benefits. Identified semaphorins/collapsins to be markedly increased upon exposure to LED that may in turn decrease pain. These cell death-associated genes were down regulated upon LED treatment in the mouse model, which suggests that there is increased proliferation induced by LED.

7. Blue laser light increases perfusion of a skin flap via release of nitric oxide from hemoglobin

Intro: Decreased blood flow is characteristic of many diseases including diabetes that can lead to multiple physiological complications. Nitric oxide is one of the most important factors in local microcirculation and activates vasodilation via the cGMP-dependent pathway. NO is produced by endothelial NO synthase. Previous studies show that NO-Hb complexes are photosensitive and can release NO during irradiation. This has possible implications in treating wounds and enhancing the inflammatory response.

Methods: A low intensity (2mW) laser light beam (wavelength of 632.8nm) was used. Scans were taken in phases. The first phase (background), started 30 min after flap harvesting. During NO-Hb/saline bolus and infusion (Infusion phase), again three scans each lasting 4 min were acquired. The same scan protocol was used for the irradiation period (Irradiation phase) as well as for the period after irradiation (Post Irradiation phase). Numeric results were calculated in percentage of perfusion units (%PU).

Results: When NO-Hb erythrocytes were placed in a sensing chamber, no free NO was detected. When the chamber was exposed to laser irradiation, free NO was rapidly increased until a plateau was reached. As soon as the laser was shut off, the NO levels decreased. Increasing NO-Hb alone does not increase anything but cGMP was higher in fresh blood. MAP and cGMP did not vary in rats when NO-Hb was used against CON and Sham alone.

Conclusion: The photodissociation of NO-Hb may be a mechanism underlying effects of laser irradiation. This study provides a new approach to improve local blood supply in a controlled manner, by local laser irradiation-induced NO release from NO-Hb complexes.

8. Illumination with blue light reactivates respiratory activity of mitochondria inhibited by nitric oxide, but not by glycerol trinitrate

Intro:   Studies have shown that NO inhibits the activity of mitochondrial respiration. This study aims to understand the other targets that are responsible for light therapy, more specifically, it wants to clarify if mitochondrial respiration resulting from NO concentrations that are relevant to in vivo scenarios can be recovered. It also wants to test for optimal settings and specific parts of the respiratory chain that contributes to this effect.

Methods: Respiratory parameters of rat liver homogenate (RLH) were determined. LED had the following characteristics: 629 nm (red), 1 W, 44 lumen; 530 nm (green), 1 W, 30 lumen; 470 nm (blue), 1 W, 10 lumen. Defined specific power supply conditions for each light source, normalizing irradiance intensity of 50 mW/cm2.

Results: The addition of NO caused complete inhibition of mitochondrial respiration. Although light at 530 and 629 nm were less efficient, blue light was capable of restoring respiration via complex I to baseline levels (100%). Blue light also restored respiration when a substrate II compound was used (green and red light were did not result in any significant effects). When GTN was used to inhibit respiration, no light of the wavelengths used were capable of recovering to baseline levels. Nevertheless, GTN has an overall weaker impact on respiration when compared to NO (80% of NO baselines).

Conclusion: Visible light facilitates the recovery of mitochondria inhibited by NO at the levels generated under septic conditions in a wavelength dependent manner: short wavelengths are more efficient. This could be a simple new clinical approach in therapy of diseases accompanied by excessive NO production and mitochondrial dysfunction.

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

Intro: LLLT has been shown to enhance many biological processes. Many of these effects include increased blood flow, enhanced mitochondrial respiration, etc. This study aims to study the effects of LLLT on human neuronal cells.

Methods: 808-nm laser, power output of 600 mW. The measured power density passing through the plateand delivered to the NHNP cells was 50 mW/cm2. The duration of irradiation was 1 sec (energy density of 0.05 J/cm2).

Results: The group treated with the laser showed a twofold increase in ATP content 7513 (+/- 970) compared to 3808 (+/- 539). It took 10min after irradiation for noticeable changes in ATP levels.

Conclusion: This study shows that low level laser irradiation at proper power and wavelength enhances ATP production in human neuronal progenitor cells. The 50 mW/cm2 was determined to be sufficient to achieve the enhanced conditions. The same effect was seen in ischemic heart models.