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Effect of Low-Energy Gallium Arsenide (GaAs, 904 nm) Laser Irradiation on Wound Healing in Rat Skin
The effects of coherent and noncoherent light in biological tissues and cells have been the subject of many studies.1–7 Wound healing and tissue repair processes are complex series of events including clotting, inflammation, granulation tissue formation, epithelization, collagen synthesis, and tissue remodeling.8 These events play an important role in wound healing, which is a serious issue for all clinicians. Timely healing establishes tissue integrity quickly and effectively in most patients. Because the delay or pause that will occur in wound healing is important to both clinician and patient, researchers have studied physical and pharmacological substances to find an accelerating agent for wound healing.
Low-level laser irradiation has gained considerable recognition and importance in the treatment of various medical problems and pathological conditions, including wound repair processes, musculoskeletal complications, impaired microcirculation, aphtous stomatitis, exudative erythema multiforme, gingivitis, and pain control.8,9 However, controversy over the efficacy of laser irradiation in wound healing exists. Some clinical studies have shown low-energy lasers accelerate the healing of injured tissue.8,10,11 Mester et al12 demonstrated increased lymphocytic phagocytosis, wound contraction rate, healing of burns, and collagen synthesis and suggested that laser therapy results were excellent in wound healing. Conversely, some researchers report that low-level laser treatment produces no beneficial effects on the wound healing process in both clinical and experimental studies.9,13–22 The aim of the present study was to evaluate the histological effect of low-energy gallium arsenide laser treatment on wound healing in rats.
Methods
Animals. Twenty-four male Wistar rats weighing 200 g to 250 g were used. The animals were acclimatized for 1 week to the laboratory conditions prior to experimental manipulation. The animals were exposed to a cycle of 12 h/light and 12 h/dark at a room temperature of 25oC. The rats had free access to standard laboratory diet and water ad libitum. The Mersin University Medical Faculty Institutional Animal Care and Use Committee reviewed and approved the procedures.
Surgical procedure. Anesthesia was induced via intramuscular injection of ketamine hydrochloride (Ketalar, Eczacibasi Ilaç Sanayi ve Ticaret AS, Istanbul, Turkey) 50 mg per kg of body weight. After anesthesia was given, the animals were placed face down on a heated mat. After shaving and sterilizing the caudal area of the back, identical wounds (5 mm diameter) were punched bilaterally in the epidermal and dermal layers of the skin. All animals were housed in individual cages specially designed to prevent bedding from entering the wound. The animals were randomly subdivided into 3 groups. In each group, 8 rats were randomly selected for laser irradiation of the right-side wound. The left-side wound was left untreated and served as control.
Laser. Low-level gallium arsenide (GaAs) laser (Petas Professional Electronics Inc, Ankara, Turkey) was used to evaluate the effects on wound healing and tissue repair processes. The laser parameters were as follows: wavelength, 904 nm; spot size, 0.28 cm2; pulse duration, 220 ns; peak power per pulse, 27 W. The irradiation parameters were as follows: pulse repetition rate, 16, 128, and 1000 Hz; average power, 0.024–0.76 MW; total daily exposure time, 15 min; delivered energy, 0.086 J, 0.68 J, and 5.31 J. The total applied energy density was 0.31 J/cm2, 2.48 J/cm2, and 19 J/cm2. Irradiation parameters were entered and automatically controlled by the laser equipment.
Irradiation. Group 1, Group 2, and Group 3 were irradiated with 0.31 J/cm2, 2.48 J/cm2, and 19 J/cm2 GaAs lasers, respectively. The wound on the right side of each animal received laser irradiation, and the left-side wound served as control. Laser irradiation of wounds was initiated the day following wound creation and continued for 7 consecutive days. The laser beam incidence angle was kept perpendicular to the irradiation surface.
Each wound was examined daily for signs of bleeding and infection.
Histological observations. The animals were sacrificed by high-dose anesthetization 21 days post injury. The bilateral wounds were immediately excised and placed in 10% neutral formaline. Routine tissue processing for light microscopy was performed on all specimens. The skin samples were embedded in paraffin. Serial sections (5 mm) were cut by microtome and stained with hematoxylin and eosin (H&E) to assess the wound healing process, leukocyte infiltration, and fibroblast count. Masson’s trichrome was used to evaluate the position of connective tissue elements in this process. Slides were examined using an Olympus BX50 light microscope (Olympus Inc, Center Valley, Pa) and photographed with an Olympus PM10SP photograph system.
During the evaluation, wound repair areas were histologically graded in a blinded fashion using a modified numerical scale.23 Inflammatory cell infiltration and fibroblast proliferation were graded (0 = absent, 1 = occasional presence, 2 = slightly distributed, 3 = abundant).
Statistical Analysis
Statistical analysis was performed using a commercially avaliable software package, Statistica 6.0 (StatSoft®, Tulsa, Okla). Differences in histological grading were evaluated according to the Kruskal-Wallis test, and multiple comparisons were performed using the Dunn test. This data were expressed as medians and 25% and 75% quartiles. In the tests, P < 0.05 was considered significant.
Results
Histological evaluation of the rat skin wounds revealed differences in wound status between the laser-treated and control wounds.
Control slides revealed normal wound healing on Day 21 (Figure 1). The healing tissues differentiated into normal skin structure. Although epithelization was incomplete, a keratin layer had formed, and the epidermis covered the defect and restored integrity to the damaged skin. The dermis was visible. Collagen bundle formation was almost complete, and skin appendages were normal (Figure 1). The inflammatory cells had almost completely disappeared.
Defect areas in laser-irradiated rats were larger than those of control wounds (Figures 2–4). The stage of epithelization was similar to the control wounds, but there were some irregularities at the superficial epidermis layer (Figure 2A). Additionally, new epidermis formation did not occur in the laser-treated group with the 19 J/cm2 laser (Group 3). A fibrin coat was detected over the defect area, and the defect was filled with granulation tissue (Group 3, Figure 4). Fibroblastic activity was prominent in the control wounds of Group 1 and Group 2 (Figures 1–3). Although collagen synthesis was observed in all laser-treated groups, the new collagen bundle formation appeared to lag behind that seen in the defect area of the control wounds (Figures 2–4). Skin appendages, especially hair follicles, were degenerated at the defect area of laser-treated wounds (Figure 2A, 3A, and 3B).
Histological grading for wound healing was evaluated according to the modified Erlich-Hunt numerical scale, including the infiltration of inflammatory cells and fibroblast count. In Group 2 and Group 3, significantly more inflammatory cells were found compared to control (P = 0.004 and P < 0.001, respectively). Fibroblast counts were found to be significantly reduced in Group 3 compared to control (P = 0.009, Table 1). In Group 1 and Group 2, the fibroblast count was reduced but not significant (P = 0.609 and P = 0.403, respectively).
Discussion
This study evaluated the effect of different doses of GaAs laser on wound healing. Many studies investigated the effect of different laser doses.24–27 The usual dose ranges are 0.1–20 J/cm2. Generally, a laser wavelength of 600 nm to 984 nm is used in physical medicine. A wavelength of 632.8 nm for a helium-neon laser and 904 nm for a GaAs laser are used most frequently in wound healing.22 The authors’ study used a GaAs laser with a wavelength of 904 nm and energy densities of 0.31 J/cm2, 2.48 J/cm2, and 19 J/cm2.
Wound healing is comprised of a continuous sequence of homeostasis, inflammation, granulation, contraction, epithelization, and repair and remodeling with connective tissue deposition. Each component of the wound healing process plays a key role through several mediators. Inflammation is the normal acute reaction of tissues after any injury and occurs through the action of neutrophils, macrophages, and lymphocytes mediated by growth factors and proteases. Proliferation takes place through fibroblasts and epithelial and endothelial cells and is largely dependent on growth factors and collagen deposition, which is an essential matrix for wound healing. Finally, remodeling is provided by collagen cross linking.
Although previous reports have suggested that laser irradiation stimulates cell proliferation, their effect on wound healing is controversial.12,17–22 The biological effects of laser irradiation may be related to wavelength, laser dosage, and laser type (direct current [19 J/cm2] or pulsed wave [0.31 J/cm2, 2.48 J/cm2]). Different procedures have been used; thus, the results are controversial. An in-vitro experiment by Lyons et al17 demonstrated that HeNe irradiation increases collagen synthesis and massive transformation of fibroblasts into myofibroblasts. Lyons et al17 also reported accelerated healing in an incisional model, and Kana et al18 found significant differences in an excisional model. Mester et al12 demonstrated positive results using both models. Others reported no difference in the rate of wound healing or collagen repair between the HeNe laser irradiation group and control wounds.17,19,20 Additionally, Allendorf et al19 and Braverman et al22 reported negative results. Surinchak et al16 demonstrated increased wound healing in early stages, but the findings were not clinically significant. Clinical observations have suggested that low-energy lasers, mainly helium-neon lasers, might stimulate wound healing. However, In de Braekt et al15 and Petersen et al13 used the GaAlAs laser (830 nm) and Anneroth et al9 used the GaAs laser, and none of the investigators found an effect on wound healing. Bouma et al25 used a GaAs-laser (904 nm) at energy densities of 0.3 J/cm2, 3.0 J/cm2, and 9.0 J/cm2 and found that the low-energy laser did not affect the inflammatory function of human monocytes and endothelial cells in vitro. Malm and Lundeberg26 used a 904 nm GaAs laser and found it did not affect venous ulcer healing in humans. Pogrel et al27 found that the GaAs laser did not have stimulatory effects on fibroblast and keratinocyte cultures. The current study used the GaAs laser and observed a normal wound healing pattern in control wounds, while impaired collagen synthesis, tissue repair, and damaged skin appendages were observed in laser-treated wounds. An increase in leukocytic infiltration (0.31 J/cm2) was found but was not significant. This increase was significant when GaAs laser doses of 2.48 and 19 J/cm2 were used. In addition, a significant decrease in fibroblast count was found when using a GaAs laser dose of 19 J/cm2. These findings suggest that the high dose of GaAs laser irradiation may cause impaired wound healing, and some functional disorders may result from skin appendage degeneration. These findings are consistent with the results using low doses of the GaAs laser.9,13,15,25–27 The results of the current study contradict those of Demir et al. Demir et al24 used a 904 nm GaAs laser at an energy density of 1 J/cm2 and found that the laser treatment was beneficial during the inflammation, proliferation, and maturation phases of healing. Demir et al observed the effects at Day 4 and Day 7 post surgery. The current study observed wound healing at Day 21 post surgery.
Conclusion
The current study findings suggest that high-dose GaAs laser irradiation negatively affects the normal wound healing process.
