Stimulation burn healing using 790 nm diode laser in rabbit ' s skin

Burn is one of the most important conditions in veterinary and human medicine. The purpose of this study is to evaluate the enhancing therapeutic effect of 790 nm diode laser on the burn healing. Twenty-five rabbits (mean weight was 2.3 ± 0.03 kg) were divided into 5 groups (A, B, F, H and control group), each group consists of 5 animals. All groups were inflected with burn (scalds) by 99 degree of Celsius hot water applied for 30 seconds, on skin of the femoral region. Four groups were treated by laser and one group was without laser irradiation as a control group. The animals were treated by diode laser five times a week at different power density. The power density (PD) of 58.9 mW/cm2 with exposure time 120 seconds was used for group (A), but in group (B), the same PD used with exposure time of 60 seconds, for group (F) the PD was 255mW/cm2 and for group (H) the PD was 888 mW/cm2 and same exposure time of 20 seconds for both last 2 groups. Results of four groups were compared with control group clinically and histopathologically. The study showed a good response in burn healing in (F) and (B) and (A) group, with no differences between group (H) and control group. The treatment of 5 times a week gave benefit in acceleration of the burn healing. The histological examinations revealed an increase in the proliferation of epithelial cells from the burn edge and decrease in the inflammatory cells infiltration in group F, B and A .The statistical results showed that the best results are in group (F) and group (B).The diode laser is effective in accelerating burn healing if used 5 times a week.


Introduction
For approximately forty years, light in form of low-level laser therapy (LLLT) has been used in treatment of a myriad of conditions. Low level laser therapy used in photobiostimulation, leading to biological and physical effects has been reported. Low level laser therapy has been found to modulate various biological processes (1), such as increasing mitochondrial respiration and ATP synthesis, facilitating wound healing (2) and promoting the process of regeneration (3).The LLLT action depends on wavelength, power density and exposure time. The most effective irradiation is that in the red and near infrared range of the spectrum. The most commonly used sources are the helium-neon laser and diode laser. In clinical practice LLLT could be considered for short-term relief of pain and treatment of many cases like wound injuries and burns, which were found to be highly, affected with LLLT (4).In this study, the effect of low level laser irradiation on burn healing in rabbits was investigated. The burns were irradiated by 790 nm diode laser in continuous mode. An understanding of the mechanism of laser tissue interaction is the basis for further development of diagnostic and therapeutic application. The purpose of this study was to evaluate enhancement effect of burn healing by using 790 nm diode lasers.

Materials and Methods:
Animal group: Rabbits were divided into five groups; each group consisted of five animals. Each group was caged separately, with standard animal pellets and diet, and was subjected to same environmental conditions. Hair plucking was made after using tranquillizer (acepromazine maleate 2) with o.5 ml/10kg body weight dose. This was done one hour before animal exposure to experimental work. General anesthesia was used to prevent animal's pain and movement. A dose of 0.24 ml/kg body weight (b.w) of xylazin 2% and ketamine HCL (in dose 0.2 ml/kg b.w) were used to anaesthetize the animal by intramuscular injection. In 4 groups the experimental burn was treated with laser with different power densities .
Burning procedure: The experiment involved two preliminary studies to determine the optimum burn type (scalds) according to the temperature achieved and the time interval of the contact, with 99 degree of Celsius hot water applied for 30 seconds. Deep second degree burns were induced figure (1). Burn wound was inflicted on dorsal surface of femoral region, by using a plastic cylinder applied perpendicularly with a 1.2 cm diameter round end (area 1.13cm²). The hot water applied directly on prepared skin segment, thus the maximal size of the burn induced was (1.3-1.4cm) in diameters .
Irradiation procedures: The rabbit was fastened on a special table by belts to prevent animal's movement during laser irradiation process and to ensure that laser light is incident perpendicular on the scalded surface and surrounding healthy skin. Burn wounds were exposed to first laser radiation after six hours postoperative, five times a week for four weeks. Thus, the healing of irradiated wounds was compared with that of non-irradiated burns in the control group. Power density and exposure time used in this work are listed in table (1). .

Measurement techniques:
Visual data were recorded every day by means of following criteria e.g., color, shape, and surface. Size index, (two-dimensional diameters of each wound) was measured by geometrical ruler each day as shown in figure (2 ( .

Figures (2) Measurement technique
Specimens for histopathological examination were prepared by taking tissue samples from the burn site of all groups, on 5th and 7thday postoperatively Results Clinical results: Clinical observations were followed and results are shown in table 2. The appearance of crust was in irradiated groups earlier than in control group. The thickness of this crust was more in control group than in irradiated groups. The crust fell down earlier in irradiated groups .

Microscopic results:
Control group: At 5th postoperative day the microscopic examination showed that epithelial cells and their nucleus are enlarged in size (see figure 7) with necrosis, destruction and sloughing of the epidermis in the burn region. The burn extended to the epidermis and dermis causing coagulation of the collagen fibers. Severe inflammatory cell infiltration (neutrophils and macrophages) are seen in the dermis and epidermis, with congestion in the blood vessels as shown in figure (8) with edema, proliferation of the fibroblasts, immature collagen fibers in dermis. The epithelialization process was began and showed layer of 1-2 epithelial cells. These cells are formed from the edge of the burn region. Epithelial cells and myofibroblasts extended to burn surface. At 7th day postoperatively, severe fibroblasts proliferation in different directions and shapes with immature collagen fiber production in the dermis are seen with severe inflammatory cells infiltrations (neutrophil, acidophil, macrophages and lymphocytes) in the dermis layer. Coagulation in collagen fibers under the epidermis with neutrophil cells infiltration is seen. Organization in the coagulated epidermis is noticed by large number of fibroblasts. The epithelial cells proliferated from the edge of healthy tissues to the burn region and formed layer of 2-3 cells with myofibroblsts. Group F: At 5th day post laser therapy, congestion in the blood vessels and mild inflammatory cells infiltration (lymphocytes, neutrophil, and macrophages) in dermis layer are seen. The epithelial cells are proliferated under epidermis region and formed a layer of 6-7 cells with myofibroblsts ( figure 9).The histological examination at 7th day of postoperative showed:-Normal appearance of the skin with complete healing of epidermis and dermis layers with all skin appendages is noticed. The hyalinization of the collagen fibers is not appearing. Group H: At 5th day post laser therapy: Severe inflammatory cells infiltration (neutrophil, acidophil, macrophages) in dermis layer and hypodermis and coagulation in collagen fibers, with congestion in the blood vessels are seen. The epidermal cells recognized by coagulation, necrosis, sloughing, and destruction and severe inflammatory cells infiltration (neutrophil, and macrophages) and invaded by fibroblasts and fibrous connective tissue production. Proliferation the epithelial cells from the burn edge and form a layer of 1-2 cells figure (10)

Figure (12) Proliferation the epithelial cells which consist of a layer of 3-5 cells, 7 day post-irradiation in Group
A.X 200, H&E.

Figure (13) Maximum burns wound area, range of mean and median of burns area.
The statistical results in all groups can be summarized in  where R means correlation coefficient, R²means determination coefficient, F means goodness of fit of mathematical models, SIG means significant degree, time B means the effect of the time in healing process, t-value means test statistical grad which null by standardized value of parameter estimation by the effect of time.
The method used in statistical results was autoregressive AR (1)-lag (1) (table3). It shows that: Correlation coefficient (R) which refers to the evaluation of the effect of time in the healing processes. And the best result was in group B, F and A respectively. And there are no differences between group H and control group. Determination coefficient (R²) was the best result in group B and F respectively and there were no differences between group A, H and control group. T-value was highest in group B and F, but there were no differences between group A, H and control group.

DISCUSSION AND CONCLUSIONS
In this study the stimulation of burn healing by laser irradiation showed acceleration healing in different stages and there is rapid healing of the burn in group F, B, A and there was no differences between group H and control group. The cause of enhancement of healing, first of all, is the increase of the proliferation of the epithelial cells, fibroblasts and new vessels formation following laser irradiation (2) . This study revealed that burn healing in group F, B and A is vastly enhanced and takes dramatically less time to complete. Also stimulation is the function of immune cells and the lymphatic and vascular systems. Where Yu (3) revealed in histological evaluations that laser irradiation improved wound epithelialization, cellular content, granulation tissue formation, and collagen deposition in laser-treated wounds and this agrees with Thamer (6) , Schindl (7) , Bisht (8) and Reddy (9). In the present work the histopathological examination revealed that the proliferation of the epithelial cells increased in group (F),group (B) and group (A) more than in group H and control group as show in figure(9),(10) and ( 12 ).these results were confirmed with Koutn study (10) where revealed that the laser light of the near-infrared region (830 nm) stimulates cell proliferation to a varying degree according to the irradiation doses and revealed at 96 hours the proliferation activity was significantly higher in the treated populations than in the non-treated cells. Mary Dyson (11) revealed that LLLT can help wounds healing. Acute inflammation is resolved more rapidly and the proliferative phase of healing begins earlier. So the low level laser therapy decreased the inflammatory reaction of wound healing and this agrees with the result of this study that indicates good response of laser therapy in group (B), (F) and confirmed the statistical results as shown in values of correlation and determination coefficient and t-value, which were more in group F and B than another groups. The good response of group F, B may be related to stimulation of the inflammatory cell or activation of the chemotactic factor by irradiation with these doses. Where Petrova (12) observed that under LLLT the high phagocytic activity of macrophages was observed as early as 6 hours after trauma. Bisht et al (8) showed an increased leukocytic infiltration and neovascularisation was seen in the laser irradiated wounds. These results participated in rapid debridement of the wound and prepared conditions for the proliferation phase. Kesava et al. (13) showed that good response may be related to changes the permeability of membrane. These physiological changes affect a variety of cells which produce the various cell types including macrophages, fibroblasts, endothelial cells and mast cells. So increase in the permeability of the cell membrane to calcium (Ca + ²) ion lead to Ca+² ion entering the cell, activating variety of enzyme molecules and can produce a cascade of intracellular signals that initiate acceleration or inhibition of biological processes ( 10) ( 15) . A cascade of reactions connected with alteration in cellular homeostasis parameters (pH, Ca+², cAMP, ATP and some others) is considered as a photosignal transduction and amplification chain in the cell , ( 1) and so that Cytochrome c oxidase is considered as a photoacceptor when cells are irradiated with monochromatic red to near-IR radiation where cytochrome c oxidase becomes more oxidized which means that the oxidative metabolism is increased due to irradiation at laser irradiation 633, 670, and 820 nm wavelength. The evidence showed that changes in the redox properties of the respiratory chain components following photoexcitation of their electronic states (16) and this supports the study of Karu (1) that revealed the mechanism of low-power laser therapy at the cellular level is based on the electronic excitation of chromophores in cytochrome c oxidase which modulates a redox status of the molecule and enhances its functional activity. Chukuka (17) and Bill (18) have shown that light energy is absorbed by endogenous chromophores in the mitochondria and used to synthesize ATP. The resulting ATP is then used to power metabolic processes; synthesize DNA, RNA, proteins, enzymes, and other biological materials needed to repair or regenerate cell and tissue components, rapid mitosis or cell proliferation and restore homeostasis. The clinical benefits resulting from these demonstrated effects are pain control and tissue repair in the multiple of circumstances. The dose in group H of laser therapy causes prolong time in the inflammation stage, may be due to inhibition in the biological processes of the cell (19) . Lagan et al (20) revealed that LLLT provides no advantages in the management of minor postoperative wounds over current practice, in this study five times laser therapy every day in group B and F revealed that the hyalinization is reduced. This results enhancement of the acceleration of wound healing in those two groups before group A, H and control group and rapid complete burns healing. The histopathological examination in group A, H indicated that the hyalinization of the collagen fibers still appear, which is related to fibroblasts proliferation. There are many studies that revealed with use of LLLT the proliferations of the fibroblast increased. Kreisler (21) , Vinck (22) revealed that when the cells are irradiated by 809-nm diode laser, a considerably higher proliferation activity will appear than the controls, where Asencio-Arana (5) showed that The differences were significantly up to 72 hours after irradiation, but Pereira (23) revealed that the Irradiation with 3 and 4 J/cm² lead to increased procollagen synthesis of cultured fibroblasts by a low-power laser. .Almeida-Lopes (24) revealed that the LLLT acts by improving in vitro fibroblast proliferation. Where Efendiev et al (25) recorded the same result with LLLT, but with increasing the power of radiation, the reparative processes in a wound are slowed and disturbed. The rate of wound contraction was proportional to cell number, that begin from the healthy tissue surrounding the wound and often increasing its surface area (26). There are many studies showed that Diode laser enhancement healing process (27), (5) (28). Where Rodriguez (29) (30) revealed that when patients whose submitted to low-intensity infrared laser beam 830 nm the healing process was faster with reduction of sore area and immediate relief of pain following first irradiation 1-The diode laser is good therapy for enhancement of tissue regeneration and burn healing. 2-The best parameter of diode laser (790 nm) enhancing burn healing was Power density 0.255 W /cm² with exposure time 20 second and Power density 0.0589 W /cm² with exposure time 60 second. 3-Increase the Power density 0.888 W /cm² with exposure time 20 didn't show any improving to burn wound healing. 4-The histological examination revealed increase the proliferation of the epithelial cells