A study of the effect of low-intensity laser radiation of the blue, green, and red spectral regions on the healing of experimental skin wounds in rats

The effect of low-intensity laser radiation of the blue (441.2 nm), green (532 nm), and red (632.8 nm) spectral regions on the healing of experimental skin wounds in rats has been studied. The effect of the traditionally applied laser radiation in the red region has been compared with the effect of laser radiation in the other spectral regions, assuming that, upon irradiation of wounds by lasers emitting in the blue and green regions, a similar effect can be achieved at lower doses. The following parameters characterizing the healing of experimental wounds were used: the functional activity of phagocytes of wound exudates, which was determined by luminol-dependent chemiluminescence, and their number; the antioxidant activity of wound exudates; and the rate of healing, which was determined as a change in the wound area. It was shown that irradiation with laser accelerated the healing of wounds in all cases. The exposure to laser radiations in the red (1.5 J/cm), blue, and green (0.75 J/cm2) spectral regions shortened the time of wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes after the exposure increased on day 5 after the infliction of the wound, whereas in the control it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after the operation. A maximum increase in the superoxide dismutase activity occurred after the exposure to laser radiation in the red region at a dose of 1.5 J/cm and in the blue and green spectral regions at a dose of 0.75 J/cm2.     

The effect of low power laser radiation of blue,green, and red ranges on free radical processes in blood of rats with experimental endotoxic shock

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was induced by intraperitoneal injection of lipopolysaccharide B (25 mg/kg) (LPS). Functional activity of blood leukocytes was evaluated by the method of luminol-dependent chemiluminescence, plasma superoxide dismutase activity was determined by the nitro blue tetrazolium assay, intensity of lipid peroxidation in erythrocyte membranes was estimated by cis-parinaric acid fluorescence. It was found that the low power laser radiation significantly influenced all investigated processes, in LPS-treated and control animals. The most pronounced effects were observed in all groups of LPS-treated animals, in which the laser radiation increased all investigated parameters. At the radiation dose 0.75 J/cm² green laser was the most effective, while at the dose of 1.5 J/cm² both green and red lasers produced potent effects. Possible mechanisms of the observed phenomena are discussed.     

The role of endogenous porphyrins in laser therapy of experimental skin wounds

The role of endogenous porphyrins in the effect of laser irradiation on the superoxide dismutase (SOD) activity of wound exudate and rat leukocyte activity has been studied on models of aseptic incised skin wounds. Wounds were irradiated with a He-Ne laser (632.8 nm, 1.5 J/cm²) on the 2nd, 3rd, and 4th days after the beginning of the experiment. Irradiation effects were evaluated by the SOD activity (NBT test) and the activity of leukocytes of the wound exudate (as a chemiluminescent response to opsonized zymosan). It was found that in animals subjected to laser irradiation, the SOD activity sharply increased. This effect depended on endogenous porphyrin concentration and was retained throughout the experiment. The SOD activity in unirradiated animals decreased from the 2nd to the 5th day of experiment. The evaluation of the activity of wound exudate leukocytes did not reveal any distinct dependence of the effect on the concentration of endogenous porphyrins.     

A comparative study of the effects of laser and light-emitting diode radiation on superoxide dismutase activity and nitric oxide production in rat wound fluid

The effect of laser and light-emitting diode radiation in the visible region of the spectrum on the content of reactive nitrogen species and superoxide dismutase activity in rat wound fluid was studied. The efficiency of action of coherent laser radiation and incoherent light-emitting diode radiation in the red region of the spectrum on the parameters analyzed was compared. The study was performed using the model of cut aseptic wounds proposed by L.I. Slutskii. A He-Ne laser (632 nm) or an U-332B light-emitting diode (630 nm) was used as the source of radiation. It was shown that (1) exposure of wounds to visible light of both laser and light-emitting diode causes dose-dependent changes in superoxide dismutase activity and nitrite production and that (2) radiation coherence does not play a significant role in the changes in superoxide dismutase activity or nitric oxide production by wound fluid phagocytes.     

Healing of burns after treatment with 670-nanometer low-power laser light

Recent investigations have reported contradictory results on the influence of low-power laser light on wound healing. Low-power laser with a power output of 250 mW and an emitted laser light of 670 nm have been insufficiently investigated to date. The effect of a 250-mW/670-nm laser light on the healing of burning wounds in rats was investigated. Thirty rats were burned on both flanks. One wound was irradiated with 670-nm laser light (2 J/cm2), whereas the other side remained untreated. Macroscopic evaluation of the wounds was performed daily; 10, 20, and 30 days after burning, 10 rats were killed and the wounds histologically evaluated. Neither macroscopic nor histologic examination of the irradiated wound showed accelerated wound healing when compared with control wounds. In the present study, irradiation of burns with a 250-mW/670-nm laser light produced no beneficial effects on wound-healing processes.     

Effect of photosensitizers pheophorbide a and protoporphyrin IX on skin wound healing upon low-intensity laser irradiation

The effect of photosensitizer with subsequent He-Ne (632.8 nm; 3 mW/cm²) laser irradiation on experimental skin wound healing has been studied. Pheophorbide a and protoporphyrin IX were used as photosensitizers. It was found that application of the photosensitizer and subsequent laser irradiation, first, decreased the amount and the functional activity of leukocytes in the wound exudate and, second, inhibited the SOD activity as compared with that of the control group. Moreover, pheophorbide and protoporphyrin practically did not affect the total healing period but decreased the length of the inflammation stage. It was supposed that these effects are related to generation of reactive oxygen species during irradiation.     

A comparative study of the effects of laser and LED radiation on lipid peroxidation in rat wound fluid

The effect of laser (632 nm) and LED (630 nm) on lipid peroxidation in rat wound fluid (exudate) was studied with the aim of comparing the efficiency of coherent and incoherent light on the processes that take place during wound healing. The study was performed using the model of cut aseptic wounds proposed by L.I. Slutskii. It was shown that irradiation of wounds with light of both laser and LED caused a decrease in the concentration of lipid peroxidation products in wound fluid as compared with the control group. An increase in the antioxidative activity of wound fluid was observed. It was concluded that irradiation with light of both laser and LED decreases the level of oxidative stress in wound fluid and that radiation coherence does not play a significant role.     

A comparison of the effects of laser and light-emitting diodes on superoxide dismutase activity and nitric oxide production in rat wound fluid

The action of laser and light-emitting diode radiation in the visible region on the content of reactive nitrogen species and activity of superoxide dismutase in rat wound fluid was studied, and efficiency of action of coherent laser and incoherent light emitting diode radiations in the red region of the spectrum on the parameters under study was compared. A model of incised aseptic wounds in rats proposed by L.I. Slutskiy was used. A He-Ne laser (632 nm) and a Y-332B light emitting diode served as radiation sources. It was shown that (1) exposure of wounds to the visible light of both laser and light-emitting diodes causes dose-dependent changes in superoxide dismutase activity and production of nitrites and (2) the radiation coherence does not play any significant role in the changes of superoxide dismutase activity or nitrogen oxide formation by wound fluid phagocytes.     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e6) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e6 demonstrated maximal effects on blood SOD activity.     

The role of tetrapyrrol photosensitizers in photoinduced variation of free radical characteristics of rat blood in endotoxic shock

The main goal of the present study was to evaluate the comparative effectiveness of tetrapyrrol photosensitizers (protoporphyrine IX and chlorine e ₆) in red (632.8 nm) and green (532.5) spectrum bands on rat blood free radical status, using the experimental model of endotoxic shock. Endotoxic shock was produced by intraperitoneal injection of lipopolysaccharide B. Irradiation effectiveness was estimated by leukocyte activation (measured with luminol-dependent chemiluminescence), superoxide dismutase activity of blood plasma (nitro blue tetrasolium assay) and lipid peroxidation (assay with cis-parinaric acid). It was found that laser irradiation has multidirectional effects on leukocyte activation, membrane lipid peroxidation and plasma SOD activity and all these effects were more pronounced in the case of endotoxic shock. Protoporphyrin was more effective in leukocyte activation and chlorine e ₆ demonstrated maximal effects on blood SOD activity.     

Tissue Responses to Postoperative Laser Therapy in Diabetic Rats Submitted to Excisional Wounds

In a previous study about low-level laser therapy biomodulation on a full-thickness burn model we showed that single and fractionated dose regimens increased wound healing and leukocyte influx similarly when compared with untreated control. In order to verify if this finding would be similar in an impaired wound model, we investigated the effect of single and multiple irradiations on wound closure rate, type of inflammatory infiltrate, myofibroblasts, collagen deposition, and optical retardation of collagen in diabetic rats. Female Wistar rats in the same estrous cycle had diabetes induced with streptozotocin and an 8-mm excisional wound performed with a punch. The experimental groups were: control group – untreated ulcer; single-dose group – ulcer submitted to single dose of diode laser therapy (λ = 660 ± 2 nm; P = 30 mW; energy density: 4 J/cm²) and fractionated-dose group – ulcer submitted to 1 J/cm² laser therapy on Days 1, 3, 8, and 10. The ulcers were photographed on the experimental days and after euthanasia tissue samples were routinely processed for histological and immunohistochemistry analyses. Independently of the energy density, laser therapy accelerated wound closure by approximately 40% in the first three days in comparison to the control group. Laser therapy increased acute inflammatory infiltrate until Day 3. Both laser groups exhibited more myofibroblasts and better collagen organization than the control group. The findings demonstrate that low-level laser therapy in the immediate postoperative period can enhance the tissue repair process in a diabetes model. Similar effects were achieved with laser therapy applied a single time with an energy density of 4 J/cm² and applied four times with an energy density of 1 J/cm². The application of laser therapy in the inflammatory phase was the most important factor to the enhancement of the tissue repair process.     

Superpulsed (Ga‐As, 904 nm) low‐level laser therapy (LLLT) attenuates inflammatory response and enhances healing of burn wounds

Low‐level laser therapy (LLLT) using superpulsed near‐infrared light can penetrate deeper in the injured tissue and could allow non‐pharmacological treatment for chronic wound healing. This study investigated the effects of superpulsed laser (Ga‐As 904 nm, 200 ns pulse width; 100 Hz; 0.7 mW mean output power; 0.4 mW/cm² average irradiance; 0.2 J/cm² total fluence) on the healing of burn wounds in rats, and further explored the probable associated mechanisms of action. Irradiated group exhibited enhanced DNA, total protein, hydroxyproline and hexosamine contents compared to the control and silver sulfadiazine (reference care) treated groups. LLLT exhibited decreased TNF‐α level and NF‐kB, and up‐regulated protein levels of VEGF, FGFR‐1, HSP‐60, HSP‐90, HIF‐1α and matrix metalloproteinases‐2 and 9 compared to the controls. In conclusion, LLLT using superpulsed 904 nm laser reduced the inflammatory response and was able to enhance cellular proliferation, collagen deposition and wound contraction in the repair process of burn wounds.

Photomicrographs showing no, absence inflammation and faster wound contraction in LLLT superpulsed (904 nm) laser treated burn wounds as compared to the non‐irradiated control and silver sulfadiazine (SSD) ointment (reference care) treated wounds     

Low-dose photodynamic therapy effect on closure of scratch wounds of normal and diabetic fibroblast cells: An in vitro study

Chronic wounds such as diabetic ulcers are a serious public health problem. Extensive research is needed to find new alternatives for wound treatment. Photodynamic therapy (PDT) is a non-invasive method, which has been studied for several decades to treat cancer, infections, and other diseases. PDT involves the administration of a photosensitizer compound followed by irradiation with using light at specific wavelength to produce reactive oxygen species (ROS) using molecular oxygen. It is possible that low dose photodynamic therapy (LDPDT) could improve wound healing and stimulates the cell repair process. This study we explored the effect of LDPDT on wound healing in vitro using normal and diabetic cellular wound models. The effects of different concentrations of 5-ALA and different energy densities (dark or light) on the cell viability of human fibroblast cells were studied using the MTT assay. After ascertaining the optimum parameters, a scratch wound assay was performed on both normal and diabetic cells and then cells treated with 1 and 5 μg/mL of 5-ALA at 1 J/cm² energy density. ROS production and morphological alteration of the cells were studied. The mortality of normal fibroblast cells increased with increasing 5-ALA concentration and also increasing energy density (up to 3 J/cm²). However, in diabetic cells, the mortality rate did not decrease. Diabetic cells showed increased migration and closure of the scratch compared to normal cells under similar conditions. A low concentration of 5-ALA (5 μg/mL) and low energy density of 1 J/cm² in both normal and diabetic cells gave a small increase in ROS levels compared to controls. This may explain the positive effects of LDPDT on wound healing. The findings of this study suggest that LDPDT may have a potential effect on the wound healing of diabetic wounds.     

Blue LED light exposure develops intracellular reactive oxygen species,lipid peroxidation,and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells

Abstract

The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm². The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm² exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm², respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm²).     

Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts

We investigated the biological effects of Er:YAG laser (2940‐nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm^?2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm^?2 promoted maximal cell proliferation, determined by WST‐8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post‐irradiation. Elevation of ATP level, Ki67 staining, and cyclin‐A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post‐irradiation at 6.3 J cm^?2, and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up‐regulation of 21 genes involved in heat‐related biological responses and ER‐associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real‐time PCR. Surface temperature measurement confirmed that 6.3 J cm^?2 generated heat (40.9°C post‐irradiation). Treatment with 40°C‐warmed medium increased proliferation. Laser‐induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm^?2 strongly potentiated HGF proliferation via photo‐thermal stress, suggesting potential wound‐healing benefit.      

The inhibition of mitosis in tissue culture cells by blue light

Blue light (wavelength 350-480 nm) irradiation of the early mitotic (prophase and prometaphase) tissue culture cells at the dose of 50-3000 J/cm2 delay mitosis or completely block it at the metaphase. Cell sensitivity to the near UV light (wavelength 360 nm) was few times more as compared with the sensitivity to the visible light (wavelength 400-480 nm). Mitotic cells irradiated with the green light (wavelength more than 500 nm; dose up to 7500 J/cm2) completed division normally. The effect of the blue light did not depend on the presence of phenol red in tissue culture medium. Rhodamin 123 staining did not show any changes in the mitochondrial system in the irradiated mitotic cells. Blue light irradiation with the dose enough for the induction of mitotic delay appears to be insufficient to affect the proliferation of interphase cells.     

Differential responses to different light spectral ranges of violaxanthin de-epoxidation and accumulation of Cbr,an algal homologue of plant early light inducible proteins,in two strains of Dunaliella

Unicellular green algae of the genus Dunaliella, similar to higher plants, respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light inducible proteins (Elips) in plants. These proteins belong to the superfamily of chlorophyll a/b binding proteins. Two Dunaliella strains, D. bardawil and D. salina, were compared for these two responses under light in the UVA, blue, green and red spectral ranges. In D. bardawil, the two stress responses were similarly induced under UVA, blue or red light and to a lesser extent under green light. In D. salina, a similar spectral range dependence was exhibited for violaxanthin de-epoxidation. However, Cbr accumulated only under UVA or blue light but not under green or red light. A strong synergistic effect of a low dose of blue light superimposed on red light resulted in Cbr accumulation. These results reveal strain-specific differences in spectral range requirements of the two light-stress responses. In the two strains, violaxanthin de-epoxidation is triggered under photosynthetically-active spectral ranges but at least in D. salina, Cbr accumulation appears to require a specific light signal additionally to a signal(s) generated by light stress.     

Role of the PI3K/AKT (mTOR and GSK3β) signalling pathway and photobiomodulation in diabetic wound healing

Activated phosphatidylinositol 3 kinase/Protein kinase B (PI3K/AKT) signalling with increased or reduced mTOR and GSK3β activity influences the wound repair process. Diabetic wounds, usually ulcerated, are characterised by reduced growth factors and cellular performance. The occurrence of diabetic ulcers is linked to peripheral arterial disease, neuropathy, and wound contamination. Lasers or light emitting diodes (LEDs) provide photon energy with therapeutic benefits (Photobiomodulation-PBM), and has been broadly commended to quicken diabetic wound healing. PBM is efficient in the visible red and near-infrared electromagnetic spectrum, and fluencies ranging from 2 to 6 J/cm². However, cellular and molecular mechanisms induced by PBM are not fully understood. In this review we discuss PBM and the PI3K/AKT pathway with specific focus on the mTOR and GSK3β downstream activity in diabetic wound healing.     

Effect of Light Quality on Chloroplast Replication in Spinach

Chloroplast growth and replication have been studied in spinachleaf dises cultured on sterile nutrient agar under lights ofdifferent spectral qualities. Over the intensity range 0.1-5mW cm^–2 both red (632 nm) and blue (488 nm) laser lightstimulate chloroplast division to the same extent as white light.By contrast low intensities (0.22–0.65 m Wcm^–2 ofboth white or green (525 nm) light are ineffective for chloroplastreplication but permit normal chlorophyll synthesis and greaterthan normal chloroplast growth. The large plastids of disesgrown in green light divide when exposed to high-intensity whitelight.