The effects of different doses of 670 nm diode laser on skin flap survival in rats

PURPOSE: To investigate the effects of different low-level laser therapy (LLLT) doses on random skin flap rats. METHODS: Forty Wistar rats were randomly divided in four groups. The control group (CG) was not irradiated. The experimental groups were irradiated with a diode laser 670 nm with different energies per point: group 2 (G2) with 0.06 J; group 3 (G3) 0.15 J and group 4 (G4) 0.57 J. The three groups were irradiated in 12 equally distributed points in the cranial skin flap portion. They were submitted to the irradiation during the immediate, first and second postoperative days. The necrosis area was evaluated in the seventh postoperative day. RESULTS: The CG shows 49.35% of necrosis area in the skin flap; G2, 39.14%; G3, 47.01% and G4, 29.17% respectively. There was a significantly difference when G4 was compared with CG`s skin flap necrosis area. CONCLUSION: The low-level laser therapy diode 670 nm with 0.57 J energy per point increases the survival in randomic skin flap rats.


Introduction
In plastic and reconstructive surgery, skin flap is the most frequently used method for repairing tissue loss following trauma or surgical procedures 1,2 .
Low-level laser irradiation may increase flap survival, provided that an appropriate amount of energy is delivered.Laser treatment has been reported to give good results when applied to skin flaps [3][4][5][6][7][8] , except for Smith 9 , who did not find any significant effect in the use of this therapy.
The state of the art in all areas of laser therapy indicates the necessity of parameterization studies in order to determine the effectiveness of therapeutic applications.
The current challenge for researchers in laser therapy is to find the appropriate parameters (wavelength, output power, emission mode, spot size and shape of the laser beam, length of irradiation, and applied technique) for the therapeutic application to be effective, whether at high or low power, or in photodynamic therapy [10][11][12][13][14] .
To the best of our knowledge, the evaluation of the viability of random-pattern skin flaps in the rat after 670 nm laser treatment at different therapeutic doses has not been carried out by any other researcher, and this provided the motivation for this study.
The purpose of this study was to investigate the effects of different irradiation doses of a 670 nm low-power diode laser on the viability of random-pattern skin flaps in the rat.

Methods
This Forty adult male Wistar rats (Rattus norvegicus albinus), weighing between 251 and 358g, were used in the study.
In the present study, a low-power diode laser model Physiolux Dual (BIOSET  Indústria de Tecnologia Eletrônica Ltda, Rio Claro, SP, Brazil) was used, emitting red light at 670 nm, with an output power of 30 mW and laser beam diameter of 0.028 cm 2 .Supplies included tracing paper, felt-tip pen, medical-surgical materials and a template made to ensure uniformity in the application of low-level laser.
All 40 rats were housed in individual cages in a temperature-controlled environment (21°C), on a 12:12 hour lightdark cycle, and fed standard rat chow and water ad libitum.The rats were randomized into 4 groups of 10 animals each, and the animals in the groups were subjected to the following procedures: Group 1 (G1) -A random-pattern skin flap was created and exposed to sham irradiation (control group); Group 2 (G2) -A random-pattern skin flap was created and exposed to 670 nm laser irradiation at an energy density of 2.14 J/cm 2 for 2s, delivering 0.06 J per point; Group 3 (G3) -A random-pattern skin flap was created and exposed to 670 nm laser irradiation at an energy density of 5.36 J/cm 2 for 5s, delivering 0.15 J per point; Group 2 (G4) -A random-pattern skin flap was created and exposed to 670 nm laser irradiation at an energy density of 20.36 J/cm 2 for 19 s, delivering 0.57 J per point.

Surgical procedure
The animals were anesthetized with tiletamine hydrochloride and zolazepam hydrochloride (25 mg/kg, intraperitoneally).Following anesthesia, the rats were placed on a flat surface with legs extended and their backs were shaved.The skin flap was outlined with a surgical skin marker considering the inferior angles of the scapula and the bones of the pelvic girdle as anatomical limits (Figure 1).After this procedure, the animals in the G1 group were anesthetized for 20 min, the laser probe was positioned and no irradiation was applied.The G2 group was anesthetized for 20 min and exposed to 670 nm laser irradiation at an energy density of 2.14 J/cm 2 for 2 s (0.06 J per point).The G3 group underwent the same procedure as the G2 group, but was exposed to laser irradiation at an energy density of 5.36 J/cm 2 for 5 s (0.15 J per point).The G4 group underwent the same procedure as the other groups, but with laser irradiation at an energy density of 20.36 J/cm 2 for 19 s (0.57 J per point) (Figure 4).Animals in the G2, G3 and G4 groups were irradiated immediately after the surgical procedure and on postoperative days 1 and 2. In all irradiations, the laser probe was placed in direct contact with the skin of the animals.

Method to estimate the percentage of flap necrosis
The percentage of skin flap necrosis was measured on postoperative day seven, using the paper template method described by Sasaki and Pang 16 .Viable tissue was characterized by warm, pink, soft, hair-bearing skin, while necrotic tissue was characterized by cold, dark, dry, hard, hairless skin.An individual template was made for each animal and the area of necrotic skin was traced.The tracing was cut out and weighed to the nearest 0.001 g on an electronic analytical balance.The percentage of necrotic skin was estimated using the formula: All sheets of tracing paper used in this study were produced by the same manufacturer and the cut paper pieces were weighted on the same electronic analytical balance.

Statistical analysis
The

Results
The results from the statistical analysis are listed in Table 1.According to the results, the percentage of skin flap necrosis in the G1 group varied from 39.43% to 58.54% (mean, 49.35%); in the G2 group, from 21.17% to 62.95% (mean, 39.14%); in the G3 group, from 38.42% to 61.97% (mean, 47.01%); and in the G4 group, from 14.80% to 41.57% (mean, 29.17%) (Figure 6).Comparison of results between the groups G1-G2, G1-G3 and G1-G4 was carried out, and a statistically significant difference was found between the G1 and G4 groups using the Kruskal-Wallis test (p=0.0007)and Dunn's Multiple Comparison test (p<0.001),while, for the other comparisons, no significant difference was found (p>0.05) (Table 2).

Discussion
In the present study, a rat model of skin flap necrosis was used.Although it is known that pig skin is more similar to human skin, a rat model was chosen because rats are readily available, relatively simple to maintain, inexpensive, and there are a large number of studies that have used this model.at its distal portion; in 5.7% of the flaps no significant necrosis was observed 17 .In order to obtain uniform conditions for the necrosis in the model, a plastic film F1, having the dimensions of the skin flap, was placed between the flap and its recipient bed to prevent revascularization, resulting in significant necrosis.In this study, a mean area of necrotic skin of 49.35% was found for the control group (no laser irradiation).
Kami et al.³ elevated 9 x 3-cm, caudally-based, dorsal skin flaps in rats and reported a mean skin flap necrosis of 53.81%.
In a later study, Smith 9 elevated 7 x 2-cm, caudally-based, dorsal skin flaps in rats, and also 7 x 2-cm dorsal skin flaps, perpendicular to the vertebral column, with a medial base, and spaced at 4-cm intervals in pigs; mean skin flap necrosis of 53.35% and 32.35% were reported for rats and pigs, respectively.Amir et al. 5 elevated 8 x 2.5-cm, cranially-based, dorsal skin flaps in rats and reported a mean skin flap necrosis of 51.5%.These random-pattern skin flap models resulted in skin flap necrosis similar to that observed in this study.However, in none of these studies a plastic barrier was placed between the flap and its vascular base.
The paper template method described by Sasaki and Pang 16 was used to evaluate the percentage of flap necrosis on postoperative day 7.This method was chosen due to its accuracy (error of less than 5%), ease of application, and because it is widely used in the literature, which facilitates comparison of results across studies.The evaluation of flap necrosis was performed by an observer unaware of the treatment the rats had received to avoid bias.Other investigators 3,17,18 have also evaluated flap necrosis on postoperative day 7, since this is the period of time needed for clear delineation of the necrotic area.
The experimental protocol is easily applicable and reproducible and did not lead to the death of the animals.No animals died during the experiments prior to euthanasia.
Several studies [10][11][12][23][24] have used low-level laser therapy as a means to reduce wound healing time, and to improve the condition of the tissue during the healing process. Otherstudies have also been carried out with the purpose to understand what laser-tissue interaction mechanisms are involved in the repair process [25][26][27][28] .
Laser irradiation parameters, such as wavelength, pulse frequency, energy density, power output, irradiance, total energy delibered, and application technique can affect cellular activities and, consequently, the obtained results.
The studies found in the literature dealing with this topic used lasers operating at wavelengths of 632.8 5,7,9 , 660 19 and 830 nm 3,4,6,8 , and emitting continuous light (Table 3).In the present study, the increase in survival observed in irradiated skin flaps agrees with previous studies [19][20][21][22] , except for Smith, 9 who did not find any significant effect in the use of laser therapy.
A large discrepancy is observed among energy density values reported by various authors, varying from 0.19 J/cm 2 9 to 288 J/cm 2 6 .According to the Arndt-Schultz law, low doses of irradiation accelerate cell activities while high doses of irradiation inhibit cell activities.In this study, the animals were randomized into 4 groups (G1 to G4), where G1 was not irradiated (control group) and the other groups were exposed to laser irradiation at energy densities of 2.14, 5.36 and 20.36 J/cm 2 , respectively.Only in the G4 group (20.36 J/cm 2 ), a statistically significant increase in flap survival was observed.However, our results and those given by Smith 9 and Kubota 6 apparently did not follow the Arndt-Schultz law since irradiations at energy densities of 0.19, 2.14 and 5.36 J/cm 2 did not ameliorate ischemic conditions of the skin flap, while an energy density of 288 J/cm 2 led to a significant increase in flap survival (Table 3).Therefore, it is important to consider other parameters that may affect cellular responses to laser irradiation in order to better understand the results.
In the studies where an increase in flap survival was observed after low-level laser irradiation [3][4][5][6] , the energy per point varied from 0.18 to 8.64 J, which agrees with the values found in the present study for the G4 group (0.56 J).In the only study in which the results were not favorable to laser stimulation 9 , the energy per point was 0.0825 J, a lower value than those mentioned above.In the present study, the energy per point for the G2 and G3 groups was of 0.06 and 0.15 J, respectively, and no beneficial effect due to laser irradiation was observed, suggesting that the tissue requires a minimum energy in order to be stimulated.It is possible that the energy per point was the key factor in explaining the discrepancy of the results, and confirms that laser irradiation affects biological activities of cells as described by the Arndt-Schultz law.Some authors [25][26][27] using low-power diode lasers emitting at 630, 780 and 904 nm have reported that laser irradiation increases blood flow in the microcirculation by inducing the release of nitric oxide (NO), a strong vasodilator, in the irradiated area.These authors have also reported that laser irradiation does not significantly increase tissue temperature, which makes it a useful treatment tool even during an acute inflammatory condition.
The literature indicates that photobiomodulation accelerates wound healing and promotes angiogenesis 25,26 , which can be used to justify the results reported in this paper.Some authors have suggested that free radicals are important mediators in ischemia and can lead to tissue destruction.
Free radicals can participate in chain-reactions, and cause peroxidation of cell membranes and damage to intracellular proteins, resulting in irreversible cellular injury.According to Stadler et al. 28 , low-level laser induce release of superoxide dismutase (SOD), which inhibits the action of free radicals.
Therefore, these factors may explain some of the results obtained in the present study.
More studies on this topic are needed since clinical results depend on a large number of variables, such as laser wavelength, energy density, pulse duration and frequency, length of treatment and a combination of these factors.Furthermore, the use of "energy delivered to the tissue" instead of "energy density" as a parameter for analysis may be necessary since many studies [3][4][5][6][7][8] show apparent contradictions in the results when the parameter energy density is evaluated.The World Association for Laser Therapy (WALT) 29 recommends on their website the use of energy dose delivered to the tissue as a dosimetric parameter in the treatment of pain.
In this website the standardization of laser treatment protocol is also recommended, as it can facilitate the comparison of results and the definition of a therapeutic window for the various clinical situations where the therapeutic laser can be used.

Conclusion
The 670 nm low-level laser was effective in improving the viability of randomized skin flaps in rats, when the irradiation TABLE 3 -Laser parameters used in various studies on irradiation of random-pattern skin flaps in the rat.
The effects of different doses of 670 nm diode laser on skin flap survival in rats Acta Cirúrgica Brasileira -Vol.27 (2) 2012 -161 energy was 0.57 J per point (energy density of 20.36 J/cm 2 ).
study was submitted to Research Ethics Committee of the Paulista University and approved certification is register by protocol 008/2011.The use of laboratory animals followed the ethical code for animal experimentation of the Council for International Organization of Medical Sciences, the norms of Brazilian Society of Science at Laboratory's Animal (SBCAL-COBEA) and the current national legislation about procedures to use animals experimentation (Federal Law 11,794 on October 9th, 2008).

FIGURE 1 -
FIGURE 1 -Dorsal view of the animal after shaving, showing the flap outline.

FIGURE 2 -
FIGURE 2 -Skin flap elevation and positioning of plastic barrier F1.

FIGURE 4 -
FIGURE 4 -Positioning of the animal, template and probe for laser irradiation.
difference in percentage of skin flap necrosis and the homogeneity of the groups were analyzed using the nonparametric tests Kruskal-Wallis and Dunn's Multiple Comparison for k independent samples.The significance level for rejection of the null hypothesis was set at p<0.05 (5%) for all tests.

FIGURE 5 -
FIGURE 5 -Distribution of the percentage of skin flap necrosis in rats.

FIGURE 6 -
FIGURE 6 -Mean area of necrosis in skin flaps of animals in the G1 group on postoperative day 7.

A 10 x 4
cm, cranially-based, dorsal skin flap was created in the rat according to the procedure described by McFarlane et al.15 which has been used as an experimental model for the study of necrosis and its prevention.Skin flap necrosis represented between 25% and 50% of the total area of the flap and was located

TABLE 2 -
Two-by-two comparisons performed after Dunn's Multiple Comparison test.
ns = not significant; hs = highly significant.