Experimental model for low level laser therapy on ischemic random skin flap in rats

Purpose: To develop an experimental model to be used in the study of low level Laser therapy on viability of random skin flap in rats. Methods: The sample was 24 Wistar-EPM rats. The random skin flap measured 10 x 4 cm and a plastic sheet was interposed between the flap and donor site. Group 1 (control) underwent sham irradiation with diode laser (830 nm). Group 2 was submitted to laser irradiation with diode laser (830 nm). The animals were submitted to Laser therapy with 36 J/cm2 energy density (72 seconds) immediately after the surgery and on the four subsequent days. The probe was usually held in contact with the skin flap surface on a point at 2.5 cm cranial from the flap base. On the seventh postoperative day, the percentage of necrotic area was measured and calculated. Results: Group 1 reached an average necrotic area of 48.86%, Group 2 – 23.14%. After the statistic analysis, compared with the control group, Group 2 showed a statistically significant increase in survival area (p<0.001).Conclusion: The experimental model proved to be reliable to be used in the study of effects of low level laser therapy in random skin flap in rats.


Introduction
The skin flap is the most frequent surgical procedure used in the plastic and reconstructive surgery.The main and most feared complication responsible for failure of procedures with skin flaps is ischemia.If it is durable it evolves to tissue necrosis, so causing an undesirable failure of the proposed treatment..2.4-6Therefore, a new research field using nonpharmacological agents, such as low level laser therapy, has emerged as an important agent for improving both microcirculation and neoangiogenesis [8][9][10][11][12][13][14] , to be studied as a possible method in the treatment of ischemic skin flaps.
Due to the characteristics of this kind of treatment, application facility, associated with the methodological deficiency of few experimental studies performed in animals on parameters and operation procedure, the purpose of this study was to develop an experimental model to be used in the study of low level Laser therapy on viability of random skin flap in rats.

Methods
The study was approved by Commission of Ethics in Research of Federal University of São Paulo[UNIFESP] going in the current apt legislation.

Laser
The diode laser equipment used in this study was Ibramed ® Equipamentos Médicos with 830 nm wavelength, 36 J/cm 2 energy density, power 30 mW and 0.07cm beam diameter.

Operation technique
All rats were anesthetized with tiletamine hydrochloride and zolazepam hydrochloride (50 mg/g) intraperitoneally during operation and during laser therapy sessions.After anesthesia induction, the rats were positioned on a flat surface with members extended and a digital trichotomy on their backs was performed.
Then, the planning of the flap through a plastic mould [film F-1 (poliester + poliethilene)] was performed, cut out in pattern sizes (10x4 cm) on animals backs, taking as limits the inferior angles of the scapulae and the superior bones of pelvis (Figure 1).The random skin flap with cranial base was cut by scalpel and elevated through deep fascia, including superficial fascia, panniculus carnosus, the subcutaneous tissue and skin 15 (Figure 2).After flap elevation a plastic barrier (film F1), with same dimensions (10x4 cm) was placed between the flap and its donor site 16,17 , and the suture was made with simple nylon 4-0 stitches (Figure 3) 2,3 .
After surgical procedure, Group 1 was kept anesthetized and subjected to sham irradiation with diode laser (830 nm) for 72 seconds and, for the four subsequent days, in determined time, with 36 J/cm 2 energy density.
Group 2 received laser treatment as follows: One point at 2.5 cm cranial from the flap base on each flap was marked and used the punctual contact technique (Figure 4).To ensure the uniformity and to ease making the markings, a 10 x 4 cm plastic film with one point fenestration was applied (Figure 5).The point was irradiated for 72 seconds.Laser treatment was daily performed for 5 days.After the laser therapy sessions, the animals went back to their respective cage and received commercial ration and water ad libitum.

Method for estimating percentage of necrotic area in distal portion of flaps
The percentage of skin flap necrosis area was calculated on the seventh postoperative day through the paper template method.The limit between viable tissue characterized by soft skin, reddish, warm and haired, and necrotic tissue (stiff, dark, cool and hairless skin) was demarcated on the animals 18 .
A mold of entire flap and the necrotic area was drawn and cut in transparent paper, being checked in a precision balance (+/-0.001gerror).After that, the following equation was used:

Results
The percentages of necrotic area among rats were: Group 1 (48.86%SD 3.76) and Group 2 (23.14%SD 5.43) (Table 1).The ANOVA test was used to analyze the variance and Bonferroni's correction was applied to compare groups.Compared with the control group, Group 2 showed a statistically significant increase in the survival area (p<0.001).(Figures 6 and 7).

Discussion
The augmentation of skin flap survival has been of great concern for plastic surgeons.In general, the delay procedure is said to be the most effective method of increasing survival area [9][10][11][12] .However, the delay procedure requires repeated operations and prolonged hospitalization.
Many experiments attempting to increase flap survival by pharmacological methods have been reported 4,6 .However, the effects of drugs are usually transient and unstable.Low-power lasers have been reported to have biostimulating effects.Due to the important circulation effects found as neoangiogenesis, the increasing of blood flow and fibroblasts proliferation, the research on low laser therapy in skin flaps was started 2,3,9 .
In the present study, a model of skin flaps with measurements of 10x4 cm developing ischemia in their distal portion was used 15 .Plastic film was interposed between the flap and the donor site to avoid the revascularization of the flap through donor site vessels, thus assuring homogeneous ischemia and necrosis conditions 16,17 .
The paper template method was selected to evaluate the percentage of necrotic area because it is simple and quick, and only requires transparent paper and an accurate scale 18 .
The energy density used was 36 J/cm 2 based on the research carried out by Kubota and Ohshiro 11 who observed, using the Doppler laser, a significant increase of vascular perfusion when using this dose.
In this study, the technique used was the punctual technique with contact, since this one is more efficient due to its small energy dispersion [9][10][11] .The probe was held in contact with the skin flap surface on a point at 2.5 cm cranial from the flap base.The irradiation lasted 72 seconds.
The results of this study showed that the difference between the experimental group (Group 2) and the control group (Group 1) were statistically significant.Group 2 was efficient for increasing the random skin flap viability in rats, maybe due to the enhancement of vascular perfusion.Moreover, low-level thermal effects cannot be excluded as a potential mechanism to increase vascular perfusion, since Stadler et al. 19 showed a thermal increase in rats, and thermal effects of irradiation are unlikely to explain the LLLT effect, but because they used infrared 830 nm laser, the skin color should be considered, particularly at higher flows.
Although the purpose of this study was not to measure the blood flow of the skin flap, it may suggest that the increase of skin flap viability of the experimental group may increase vascular perfusion.

Conclusion
The experimental model proved to be useful in the study of effects of low level laser therapy on viability of random skin flap in rats.

TABLE 1 -
Percentage of necrotic area in the 7 th posoperative day