Effects of low-power laser irradiation (LPLI) at different wavelengths and doses on oxidative stress and fibrogenesis parameters in an animal model of wound healing

Gallium-arsenide (GaAs) and helium-neon (HeNe) lasers are the most commonly used low-energy lasers in physiotherapy for promoting wound healing and pain modulation. The aim of this study was investigate the effect of low-power laser irradiation (LPLI) at different wavelengths and doses on oxidative stress and fibrogenesis parameters in an animal model of wound healing. The animals were randomly divided into five groups (n = 6): Controls (skin injured animals without local or systemic treatment), skin injury treated with HeNe 1 J/cm² (two seg); skin injury treated with HeNe 3 J/cm² (six seg); skin injury treated with GaAs 1 J/cm² (three seg); skin injury treated with GaAs 3 J/cm² (nine seg). A single circular wound measuring 8 mm in diameter was surgically created on the back of the animal. The rats were irradiated at 2, 12, 24, 48, 72, 96, and 120 h after skin injury. The parameters, namely hydroxyproline content, activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and lipid (TBARS) and protein oxidation (carbonyl groups) measurements were assessed. In addition, wound size regression was also analyzed. The results showed an improvement in the wound healing reflected by the reduction in wound size and increased collagen synthesis. Moreover, a significant reduction in TBARS levels, carbonyl content, and SOD and CAT activities were observed after laser irradiation, particularly with the treatments HeNe laser 1 and 3 J/cm² dose and GaAs 3 J/cm² dose. The data strongly indicate that LPLI therapy is efficient in accelerating the skin wound healing process after wounding, probably by reducing the inflammatory phase and inducing collagen synthesis.     

Effect of low-intensity laser irradiation (660 nm) on a radiation-impaired wound-healing model in murine skin

BACKGROUND AND OBJECTIVE: The use of low-intensity laser therapy (LILT) as a therapeutic modality has become popular in a variety of clinical applications including the promotion of wound repair. Although the clinical evidence base for such application remains sparse, recent studies have demonstrated a number of quantifiable photobiological effects associated with such therapy. In the present study, the effect of laser irradiation at various radiant exposures on a radiation-impaired wound-healing model in murine skin was investigated. STUDY DESIGN/MATERIALS AND METHODS: The study included two phases; in phase one, male Balb/c mice (n = 36; age-matched at 10 weeks) were randomly allocated to three experimental groups (n = 12, each group). In all groups, a well-defined area on the dorsum was exposed to 20 Gy x-rays. Seventy-two hours postirradiation, all mice were anaesthetised and a 7 x 7 mm area wound was made on the dorsum. All wounds were videotaped alongside a marker scale (three times weekly) until closure was complete. In groups 2 and 3, mice were treated with laser irradiation (0.5 and 1.5 J/cm(2), respectively) three times weekly by using a 660-nm GaAlAs laser unit (5 kHz; 15 mW; Omega Laser Systems, London, UK). Wound areas were then calculated by using an image analysis system (Fenestra 2.1), and results were analyzed by using repeated measures and one-factor analysis of variance statistical tests. In phase two, two experimental groups were included (n = 12 each group); the protocol was identical to that described for phase 1; however, mice in group 2 were treated with a radiant exposure of 4 J/cm(2). RESULTS: Results from this investigation demonstrated that treatment with 0.5, 1.5. and 4 J/cm(2) had no beneficial effect on the rate of wound closure (P > 0.05). CONCLUSION: These findings provide little evidence of the putative stimulatory effects of LILT in vivo at the parameters investigated.     

In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes

BACKGROUND AND OBJECTIVE:The effects of low-level laser light irradiation are still highly contested, and the mechanisms of its action still unclear. This study was conducted to test the effects of low-level laser irradiation at 660 nm on human lymphocytes and to investigate the possible mechanisms by which these effects are produced. STUDY DESIGN/MATERIALS AND METHODS: Whole blood obtained by phlebotomy was irradiated at 660 nm by using energy fluences between 0 and 5.0 J/cm(2). The lymphocytes were isolated after irradiation of the whole blood. For the control experiment, the lymphocytes were first isolated and then irradiated at the same wavelength and energy fluence for comparison. The proliferation of lymphocytes and the formation of free radicals and lipid peroxides were monitored. Hemoglobin was also irradiated in a cell-free environment to test for the production of lipid peroxides. RESULTS: Lymphocyte proliferation was significantly higher (P<0.05) as expressed by a Stimulation Index in samples irradiated in the presence of whole blood compared with lymphocytes irradiated after isolation from whole blood. Free radical and lipid peroxide production also increased significantly when samples were irradiated in the presence of red blood cells. CONCLUSION: The present study supports the hypothesis that one mechanism for the photobiostimulation effect after irradiation at 660 nm is the reaction of light with hemoglobin, resulting in oxygen radical production.     

Effect of low-power GaAlAs laser (660 nm) on bone structure and cell activity: an experimental animal study

Low-level laser therapy (LLLT) is increasingly being used in the regeneration of soft tissue. In the regeneration of hard tissue, it has already been shown that the biomodulation effect of lasers repairs bones more quickly. We studied the activity in bone cells after LLLT close to the site of the bone injury. The femurs of 48 rats were perforated (24 in the irradiated group and 24 in the control group) and the irradiated group was treated with a GaAlAs laser of 660 nm, 10J/cm² of radiant exposure on the 2nd, 4th, 6th and 8th days after surgery (DAS). We carried out histomorphometry analysis of the bone. We found that activity was higher in the irradiated group than in the control group: (a) bone volume at5 DAS (p=0.035); (b) osteoblast surface at 15 DAS (p=0.0002); (c) mineral apposition rate at 15 and 25 DAS (p=0.0008 and 0.006); (d) osteoclast surface at 5 DAS and 25 DAS (p=0.049 and p=0.0028); and (e) eroded surface (p=0.0032). We concluded that LLLT increases the activity in bone cells (resorption and formation) around the site of the repair without changing the bone structure.     

Effects of a specially pulsed electric field on an animal model of wound healing

The possible beneficial effects of a specially pulsed electric field (PEF) on wound healing were investigated in this study. We made a pair of triangular, full-thickness, dorsal incisions in the skin of 32 healthy male mice (one control group and three exposure groups). The treatment groups were kept between parallel plates in a partially insulated exposed environment. Group I was exposed to an electric field intensity of 10 kV/m, group II was exposed to 1.9 kV/m, and group III was exposed to 0.9 kV/m. PEFs were applied to the subjects for 20–22 h and 8 consecutive days. We determined the differences in wound recovery between the groups based on the following parameters: collagen fiber density, inflammatory infiltration density, capillary proliferation, and existence of exudates. We found that a 0.9 kV/m–1.9 kV/m chopped direct current (DC) electric field with a 30 μs repetition time favorably affected collagen synthesis and wound recovery. Despite the intensity of 0.9–1.9 kV/m, PEF accelerated healing, but 10 kV/m decelerated this recovery process.     

Excimer laser irradiation of non-calcified and calcified human atheroma (248 and 308nm wavelengths)

In order to develop a system of peripheral arterial angioplasty, we carried out an in vitro study to define the quantitative, thermal and morphological characteristics of human-atheroma ablation by excimer laser. A multigas ‘Sopra’ laser was used. The study was performed by using 248nm, krypton fluoride (KrF), then 308nm, xenon chloride (XeCl) wavelengths. The delivered energy was up to 150 mJ pulse⁻¹, pulse duration was 25ns, and the repetition rate could be adjusted to up to 20Hz. Irradiated tissue segments of the superficial femoral and external iliac arteries were obtained in man during surgical procedures and were both calcified and non-calcified atherosclerotic lesions. Quantitative measurements showed a linear increase of ablated tissue mass depending on the energy delivered. For the same energy, the loss of mass was greater with the 248nm wavelength than with the 308nm. The maximum temperature rise measured at the site of irradiation was 6°C at 248nm and 25°C at 308nm. Histological analysis of the irradiated segments revealed neat and precise ablation without thermal injury of adjacent tissue. At 248nm, this phenomenon was observed for calcified as well as non-calcified atheromas. It is concluded that quantitative, thermal and morphological characteristics of in vitro ablation of calcified and non-calcified human atheroma by excimer laser are compatible to clinical requirements. The results observed at 248nm were experimentally more satisfactory.     

Low-power laser light in the healing of burns: a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group

BACKGROUND AND OBJECTIVE: Studies on the influence of low-power laser light on wound healing have shown inconsistent results, or, as in the case of burns, are very scarce. We have studied the effects of two different low-power diode laser lights on the healing of burns in rats. STUDY DESIGN/MATERIALS AND METHODS: Thirty rats were burned on both flanks and randomly allocated to one of three study groups. In group A, both wounds remained untreated; in groups B and C, one wound each was irradiated with 635 nm or 690 nm laser light (1.5 J/cm(2)), whereas the other wound remained untreated. Diameter, redness, and edema of the wounds were examined daily. RESULTS: Between and within groups, diameter, redness, and edema of the wounds were similar throughout the entire observation period. Irradiation of the burns did not accelerate wound healing when compared with control wounds. CONCLUSION: We conclude that neither 690 nm nor 635 nm low-power laser light produced any beneficial effects on the healing processes of burns in rats.     

The effects of diode laser (660 nm) on the rate of tooth movements: an animal study

Lasers in Medical Science - Low-level laser has been indicated to have the capability to facilitate the differentiation of the osteoclastic and osteoblastic cells which are responsible for the bone...     

Measurement of thermal effects on the optical properties of prostate tissue at wavelengths of 1,064 and 633 nm

BACKGROUND AND OBJECTIVE: The extent of thermal injury during laser prostatectomy is dependent on the light distribution in laser-irradiated tissue. As tissue is irradiated, the optical properties change as a function of temperature due to an alteration of molecular and cellular structure. The purpose of the present study was to determine how the exposure of both fresh and previously frozen canine prostate tissue to elevated temperatures affects the optical properties. STUDY DESIGN/MATERIALS AND METHODS: Optical properties were measured by using a double integrating sphere spectrophotometer with an inverse adding-doubling algorithm. Measurements were made at two wavelengths (1,064 nm and 633 nm) on samples heated in a waterbath in 5 degree-10 degree increments for 10 min through a 50 degrees C temperature range. RESULTS: Upon coagulation, the absorption coefficient of fresh tissue decreased from the baseline measurement for both wavelengths (0.027 +/- 0.003 to 0.019 +/- 0.002 for lambda = 1,064 nm; 0.073 +/- 0.007 to 0.061 +/- 0.006 for lambda = 633 nm). However, the scattering coefficient increased sharply from the baseline measurement following coagulation (3.06 +/- 0.26 to 6.05 +/- 0.29 for lambda = 1,064 nm; 4.89 +/- 0.23 to 7.22 +/- 0.30 for lambda = 633 nm). Thermal coagulation occurred during exposure to temperatures between 60 degrees C and 70 degrees C. CONCLUSION: Data obtained in this study indicate that thermal coagulation of tissue alters the optical properties. The extent to which these changes occur was found to be dependent on wavelength and freshness of tissue. These results are significant because they suggest how thermally induced changes in the optical properties may limit the depth of light penetration in tissue thus compromising treatment.     

Investigation on radiofrequency and laser (980 nm) effects after endoluminal treatment of saphenous vein insufficiency in an ex-vivo model.

OBJECTIVES: An ex-vivo model for the experimental evaluation of endoluminal thermal procedures for occlusion of saphenous veins was developed. Radiofrequency obliteration (RFO) and endovenous laser therapy (ELT) were compared using this model. DESIGN: Experimental ex-vivo treatment study. MATERIALS AND METHODS: The model consists of the subcutaneous foot veins from freshly slaughtered cows which were reperfused in situ with heparinised bovine blood. The veins were treated with either radiofrequency (RFO n=5) or with endoluminal 980 nm laser light (ELT n=5) using a continuous pull-back for RFO and a stepwise illumination and pull-back protocol for ELT. Immediately after treatment perivenous tissue and veins were examined macroscopically. In a second study the same treatment parameters were used in four further vein segments with RFO (n=2) and ELT (n=2). These vein segments were examined microscopically in HE-stained histological sections. RESULTS: Induration of the vessel wall and contraction of the vessel lumen were observed after RFO. Laser treatment produced carbonised lesions of the vein wall. After 12-24 laser exposures these lesions often became transmural, causing complete perforation of the vessel wall. Histological evaluation after radiofrequency treatment demonstrated homogenous circular thermal tissue alteration with disintegration of intima and media structures. Histological evaluation after endovenous laser treatment showed large variations of thermal tissue effects. Tissue effects ranged from major tissue ablation and vessel wall disruption to minor effects located between laser exposures and on the opposite vessel wall. CONCLUSIONS: Our model is suitable for systematic scientific evaluation of endovenous thermal occlusion procedures. Our first results and theoretical considerations indicate that endovenous laser treatment should be modified in order to ensure controlled homogenous circular thermal damage, avoiding vessel wall perforation and damage to perivascular structures.     

A useful algorithm for determining fluence and pulse width for vascular targets using 1,064 nm Nd:YAG laser in an animal model

BACKGROUND AND OBJECTIVES: Many current parameters to ablate vascular beds using 1,064 nm lasers are based on high-energy settings and often fail to consider vessel diameter and/or pulse width. This study attempts to define the minimal effective dosage (MED) of energy and pulse width for specific vessel diameters in an animal model. STUDY DESIGN/MATERIALS AND METHODS: 1,064 nm Nd: YAG was used in 15 Sprague-Dawley rats. Bilateral extended dorsolateral skin flaps were elevated and vessel diameters from 0.1 to 1 mm were identified. Pulse widths (PW) in a range of 15-60 milliseconds and fluences between 70-110 J/cm2 with contact cooling at 5 degrees C (Celsius) were utilized. Results were determined clinically and histologically. RESULTS: Ideal pulse width and MED for each vessel diameter were determined using a 6 mm spot size. Histology showed early hemostasis and subsequent thrombosis, which are consistent with clinical findings. CONCLUSIONS: This model allows in vivo monitoring of vessel ablation.Optimal pulse width and MED levels versus vessel diameter determined in this animal model provide a useful algorithm that may allow for more effective treatment of vascular targets utilizing the 1,064 nm Nd:YAG laser.     

Effect of helium-neon and infrared laser irradiation on wound healing in rabbits

We examined the biostimulating effects of helium-neon laser radiation (HeNe; 632.8 nm), pulsed infrared laser radiation (IR; 904 nm), and the two combined on skin wound healing in New Zealand white rabbits. Seventy-two rabbits received either 1) no exposure, 2) 1.65 J/cm2 HeNe, 3) 8.25 J/cm2 pulsed IR, or 4) both HeNe and IR together to one of two dorsal full-thickness skin wounds, daily, for 21 days. Wound areas were measured photographically at periodic intervals. Tissue samples were analyzed for tensile strength, and histology was done to measure epidermal thickness and cross-sectional collagen area. Significant differences were found in the tensile strength of all laser-treated groups (both the irradiated and nonirradiated lesion) compared to group 1. No differences were found in the rate of wound healing or collagen area. Epidermal growth was greater in the HeNe-lased area compared to unexposed tissue, but the difference was not significant. Thus, laser irradiation at 632.8 nm and 904 nm alone or in combination increased tensile strength during wound healing and may have released tissue factors into the systemic circulation that increased tensile strength on the opposite side as well.     

The effects of a diode laser (810 nm) on pigmented guinea-pig skin

Various types of lasers, such as the Q-switched ruby laser and the Alexandrite laser, cause selective damage to cutaneous pigmented cells and are currently used in the therapy of pigmented lesions. The aim of this study was to evaluate the effects of a diode laser at a wavelength of 810 nm on pigmented guinea-pig skin. The diode laser was supplied by OcuLight Iris Medical Instruments, Inc. and was used to deliver 0.15, 0.2, 0.3, 0.4, 0.5, 1 and 1.5 J/cm² laser beams in micropulses of 100 μs. The study was carried out on albino and black-spotted guinea pigs (GP). After irradiation, punch biopsies were taken and analysed by light and electron microscopy. Albino animals developed just a few signs of cutaneous injury. This mostly consisted of spongiotic disarray, after the highest doses were administered (>1 J/cm²). In the black skin of spotted GPs alterations appeared at 0.15 J/cm² and included melanosome damage and, at doses higher than 0.3 J/cm², also melanocyte damage. These observations demonstrate that the diode laser at 810 nm selectively affects pigmented structures and that the cellular targets of diode laser radiation are the melanosomes. The diode laser specificity for melanin may provide a biological basis for the treatment of pigmented superficial cutaneous lesions. Paper received 15 December 1999; accepted 5 October 2000.     

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.