Photomedicine and Laser SurgeryVolume 27, Number 2, 2009© Mary Ann Liebert, Inc.Pp. 337–343DOI: 10.1089/pho.2008.2295

Effect of Low-Level Laser Therapy on Mast Cells in Viability of the Transverse Rectus Abdominis Musculocutaneous Flap

Carlos Eduardo Pinfildi, P.T., Ph.D.,1 Richard Eloin Liebano, P.T., Ph.D.,2

Bernardo S. Hochman, M.D., Ph.D.,3 Milvia M.M.S.S. Enokihara, M.D., Ph.D.,4

Rodrigo Lippert, P.T.,3 Rafael Corrêa Gobbato, P.T.,3 and Lydia Masako Ferreira, M.D., Ph.D.5

Abstract

Objective: To assess the effect of low-level laser therapy (LLLT) on viability of mast cells of the transverse rec-tus abdominis musculocutaneous (TRAM) flap. Background Data: LLLT has been recently used on the TRAMflap to stimulate mast cells. Materials and Methods: Eighty-four Wistar rats were randomly divided into sevengroups of 12 rats in each: group 1 (sham laser therapy); group 2 received 3 J/cm2 at one point; group 3 received3 J/cm2 at 24 points; group 4 received 72 J/cm2 at 1 point; group 5 received 6 J/cm2 at 1 point; group 6 re-ceived 6 J/cm2 at 24 points; and group 7 received 144 J/cm2 at 1 point. All experimental groups underwentLLLT immediately after TRAM surgery and on the next two following days, for three sessions in total. The per-centage of the area of skin flap necrosis was calculated on the fourth postoperative day and two samples ofskin were collected from each rat with a 1-cm2 punch to perform mast cell evaluations with toluidine blue dye.Results: Statistically significant differences were found in the percentage of necrosis, and higher values wereseen in group 1 than in all other groups. Among groups 3–7 no statistically significant differences were found(p � 0.292). For mast cells, when group 1 was compared to groups 5 (6 J/cm2 at 1 point) and 7 (144 J/cm2 at 1point), it had fewer mast cells. Conclusion: LLLT at a wavelength of 670 nm was effective at reducing thenecrotic area, and we found that it can stimulate mast cells growth to increase vascular perfusion.

337

Introduction

SINCE THE CLASSICAL STUDY of Hartrampf et al. in 1982,1 thetransverse rectus abdominis musculocutaneous (TRAM)

flap has become the preferred type of autogenous tissue touse for breast reconstruction.2–4

However, the TRAM flap is not a perfect solution due tocomplications related to abdominal wall integrity, as well asinsufficient flap perfusion, which may cause partial loss ofthe flap.5–7 These complications may lead to a need for fur-ther surgical intervention, thus increasing hospital stay anddelaying the return to activities of daily life.8–10

In an attempt to find alternatives that reduce or preventtissue necrosis, some studies using experimental modelshave tried different kinds of drugs, such as vasodilators, an-ticoagulants, antioxidants, prostaglandin inhibitors, calciumchannel blockers, and anti-adrenergic agents.9,10

The use of some of these drugs, however, has undesiredsystemic effects, increasing the need for non-pharmacologicmeans, such as acupuncture and electroacupuncture,11 low-level laser therapy (LLLT),12–15 and polarized low-frequencyelectric current.16–18 To improve the viability of flaps, the useof LLLT is being increasingly highlighted because of its easeof application and its good results at both the macroscopicand cellular levels.

In the literature, there are no studies that evaluate thenumber of mast cells found in cutaneous and TRAM flaps,but there are studies proving the importance of mast cell de-granulation in stimulating blood flow.19,20

Mast cells are IgE-sensitive, and when they come into contact,they stimulate the delivery of chemical mediators found withinmast cell vesicles (degranulation) such as serotonin, heparin, andhistamine. Both serotonin and histamine have vasoactive pro-prieties that are important in improving vascular perfusion.21

1Department of Plastic Surgery and IMES-FAFICA, 2Department of Plastic Surgery and UNICID, 3Department of Plastic Surgery, 4De-partment of Pathology, and 5 Plastic Surgery, Division of Surgery Department and Post-Graduate Program in Plastic Surgery, São PauloFederal University (UNIFESP), São Paulo, SP, Brazil.

Pinfildi et al.15 used a helium-neon laser with a 632.8-nmwavelength, and they had statistically significant results inimproving viability of skin flaps in rats. Currently, there isno consensus about the most efficient wavelength to use toincrease ischemic flap viability.

Therefore, this study was designed to assess the effect ofLLLT on mast cells and TRAM flap viability.

Materials and Methods

To perform this study, 84 adult 3-mo-old male rats (Rat-tus norvegicus) of the Wistar EPM-1 (UNIFESP) strain, weigh-ing 260–320 grams each, were used. The animals were keptin individual cages, with a normal day-night light cycle un-der controlled temperature, and were fed a standard diet andhad access to water ad libitum. This study was approved bythe UNIFESP Ethics Committee in Research.

Groups and experimental protocol

Before the experiment, all 84 rats were weighted and ran-domly assigned using Urn Randomization Software from theUniversity of Connecticut Health Center to seven groups of12 animals each.

In group 1 (sham LLLT), the animals received simulatedlaser irradiation with the device turned off. Group 2 (3 J/cm2

at 1 point) received laser irradiation with only one irradiatedspot on the TRAM pedicle, at a total energy dose of 0.18 J (6sec of irradiation). Group 3 (3 J/cm2 at 24 points) receivedlaser irradiation at 24 spots, in and around the TRAM flap,for a total energy dose of 4.32 J (2 min 24 sec of irradiation).Group 4 (72 J/cm2 at 1 point) received just one laser irradi-ation spot on the TRAM pedicle, with a total energy dose of4.32 J, also for 2 min 24 sec. Group 5 (6 J/cm2 at 1 point) re-ceived just one laser irradiation spot on the TRAM pedicle,

with a total energy dose of 0.36 J (12 sec of irradiation). Group6 (6 J/cm2 at 24 points) received 24 laser irradiation spots,in and around the TRAM flap, for a total energy dose of 8.64J (4 min 48 sec of irradiation). Group 7 (144 J/cm2 at 1 point)received just one laser irradiation spot on the TRAM pedicle, for a total energy dose of 8.64 J, also for 4 min 48sec (Fig. 1).

The technique used for the laser irradiation was contactmode, in which the laser probe remains in contact with theanimal during the entire irradiation period of each spot. Thelaser probe was kept at a 90° angle, perpendicular to the flap.

A plastic template was used to mark the sites to be irra-diated. All applications were performed in the same areason every animal. The distance between the spots and the flapborder was 1 cm in all cases.

Laser irradiation was performed immediately after sur-gery, and also on the two following days, so all animals un-derwent three applications.

Equipment

We used a diode low-level InGaAlP laser in the visible redrange (wavelength 670 nm), with a power output of 30 mW.The operator wore protective glasses, and the spot size was0.06 cm2 and the laser was used in continuous mode.

Surgical technique

To perform the TRAM procedure, the animals were anes-thetized by intraperitoneal administration of 50 mg/kg oftiletamine hydrochloride and zolazepam hydrochloride. Theresearcher who performed the TRAM procedure did notknow to which group each animal belonged. The flap was3 � 5 cm in size, and was positioned 1 cm caudal to thexiphoid appendix. The skin was cut around the flap perim-

PINFILDI ET AL.338

FIG. 1. (A) Site of laser irradiation with a single spot on the TRAM pedicle. (B) Site of laser irradiation with 24 spots, onand around the TRAM pedicle.

BA

eter. The flap dissection began on the left side of the pedi-cle, with dissection on the supra-aponeurotic plane from lat-eral to medial, until the midline was reached.

On the right side of the pedicle, the flap was dissected un-til the lateral border of the right rectus abdominis musclewas reached. The flap was then elevated by incision alongthe midline (linea alba). Elevation of the flap was completedby incision of the cranial portion and the lateral border ofthe rectus abdominis muscle, so the flap remained attachedonly by the right rectus abdominis muscle, near the rightcaudal epigastric artery.

Then the area from which the muscle was removed wasclosed with a running suture of 5-0 nylon. The skin was thensutured with 5-0 nylon, first with separate sutures for fixa-tion of the flap to the midline, then on the four edges of theflap. The closure of the skin was done with a running sutureover the perimeter of the flap.

Macroscopic evaluation of the necrotic area

The percentage of the area of skin flap necrosis was cal-culated on the fourth postoperative day using a paper tem-plate.22,23 The line between the viable tissue, characterizedby soft, warm skin and a reddish color, and the necrotic tis-sue, which was stiff, dark, and cool, was demarcated on eachanimal. A sketch of the entire flap and necrotic area wasdrawn and cut out of transparent paper, then the weight waschecked with a precision balance accurate to � 0.001 gram.Then the following equation was used to calculate the per-centage of necrosis present:

Percentage of necrotic area of the TRAM flap �weight of paper template of TRAM flap necrosis

weight of paper template � 100

of total area of TRAM flap

Mast cell histology and morphometry

Immediately after measurement, two samples of skin werecollected using an 1-cm2 punch to perform mast cell evalu-ation with toluidine blue dye to count mast cells.

The first sample (sample A) was collected ipsilateral to thepedicle (right side) of the flap, from the portion over the pedi-cle 1 cm above the flap’s caudal border. The second sample(sample B) was taken from outside the flap, 2 cm from theflap’s left caudal border (Fig. 2).

The samples were examined with an optical microscope(Olympus BH-2; Olympus, São Paulo, Brazil) at 400� mag-nification. The quantitative study was performed by two dif-ferent investigators, who using an ocular lens with 10� mag-nification, placed a checkered grid over the area to beassessed. The grid had 100 squares 0.25 mm in size and wascoupled to the microscope, so only cells within the squareswere counted. For the morphometric assessment, five areas

LOWER-LEVEL LASER THERAPY ON MAST CELLS 339

FIG. 2. Sites where skin samples A and B were taken formast-cell assessment.

TABLE 1. AVERAGE OF THE PERCENTAGE OF NECROTIC AREA AND MAST CELL VALUE IN THE STUDY GROUPS

Animal Control 3 J/cm2 1 pt. 3 J/cm2 24 pt. 72 J/cm2 1 pt. 6 J/cm2 1 pt. 6 J/cm2 24 pt. 144 J/cm2 1 pt.

01 50 20.98 12.12 9.52 1.07 9.41 5.5502 31.57 13.63 31.88 9.87 14 4.44 4.9103 28.57 19.11 11.62 2.32 13 0 004 37.77 23.61 4.30 6.89 0 4.44 4.2505 43.75 16.43 5.12 7.93 24.19 13.63 11.1106 25.56 29.03 0 5.88 0 14.28 10.8607 40 35.89 0 18.36 21.34 10 6.3808 23.58 17.33 11.62 6.66 0 8.51 8.1609 39.24 37.73 19.60 6.665.49 6.81 5.5510 44.82 13.08 0 9.90 21.12 6.66 11.5311 29.33 26.08 3.03 6.97 10.44 7.69 19.1412 56.06 22.5 15.38 9.30 5.94 10.41 13.63Average 37.49% 22.95% 9.55% 8.28% 9.71% 8.57% 8.42%Mast cells (A) 1.0 0.9 1.1 1.1 1.7 1.4 1.7Mast cells (B) 1.1 0.8 1.3 1.0 1.4 1.0 1.0

Necrosis in group 1 � groups 2–7 (p � 0.000).

of each sample were examined. The investigators did notknow to which group each sample belonged.

Statistical analysis

To determine if statistically significant differences existedamong the seven study groups in terms of percentage of ne-crosis, ANOVA was performed.

To ascertain whether statistically significant differences ex-isted among the numbers of mast cells present, the Mann-Whit-ney U-test was applied to the relative values of mast cells insamples A and B. Then the Kruskal-Wallis test was performedto check whether statistically significant differences existed be-tween the group averages for mast cells in samples A and B.

To determine whether a statistically significant relation-ship existed between the percentage of necrotic area and thenumbers of mast cells, Spearman’s correlation coefficientwas applied to the evaluations of the first investigator. Sta-tistical significance for all values was set at p � 0.05.

Results

As shown in Table 1, statistically significant differenceswere found among percentage of necrosis, with the highestvalues of all groups found in group 1. Among groups 3–7,no statistically significant differences were found (p � 0.292).

Group 1 (control) had fewer mast cells than groups 5 (6J/cm2 at 1 point) and 7 (144 J/cm2 at 1 point). Group 2 (3J/cm2 at 1 point) had fewer mast cells than groups 5 (6 J/cm2

at 1 point), 6 (6 J/cm2 at 24 points), and 7 (144 J/cm2 at 1point). Group 4 (72 J/cm2 at 1 point) had fewer mast cellsthan group 7 (144 J/cm2 at 1 point). Group 2 (3 J/cm2 at 1point) had fewer mast cells than groups 3 (3 J/cm2 at 24points) and 5 (6 J/cm2 at 1 point). Group 3 (3 J/cm2 at 24points) had fewer mast cells than groups 4 (72 J/cm2 at 1point) and 7 (144 J/cm2 at 1 point). Group 5 (6 J/cm2 at 1point) had more mast cells than the other groups.

According to results seen in Table 2 a negative correlationwas found in the mast cell evaluation, meaning that as thevalue of a variable increases, the values of the others de-crease, and vice versa. However, this was not a statisticallysignificant difference.

Discussion

The present study showed that low-level laser (InGaAlP)therapy at 670 nm improved TRAM flap viability and en-hanced mast cell numbers. One advantage of this is that mostTRAM flap complications are due to poor perfusion.7,24 Be-cause it is a pedicled flap on the right rectus abdominis mus-cle, its perfusion depends on the right caudal epigastricartery, which makes it susceptible to necrosis of the pedicle’scontralateral region.25

As flap viability is dependent on the quality of perfusion,anything that can be done to improve perfusion is desirable,and to this end LLLT was first studied in cutaneous flaps byKami et al.,26 who found significant results in terms of in-creasing flap viability.

Soon afterward, other studies were carried out, but therewas standardization of techniques, and studies such as thosedone by Smith et al.,27 Kubota and Oshiro,12 and Amir etal.,13 all found good results, but comparisons between theirresults are impossible due to the range of techniques and pa-rameters they used. In another study of this technique byPinfildi et al.,15 they found that application both inside andaround a cutaneous flap with laser energy at 632.8 nm (He-Ne) resulted in 21% of necrotic area, compared to 48% incontrols, which led to an increase of flap viability. Prado etal.14 also studied this technique, with application to only onepoint on the cranial aspect of a cutaneous flap, but they useda wavelength of 830 nm (AsGaAl), but they also found goodresults in terms of reduction of tissue necrosis.

Aside from the study by Kubota,28 in which a blood flowincrease was found after laser irradiation at 830 nm (AsGaAl)of a single site on the pedicle of an axial flap, there is a lackof studies of axial flaps and LLLT.

Thus, the utilization of LLLT in TRAM flaps is a new con-tribution to the literature. In the present study we found anaverage necrotic area of 37.49%, which is in accordance withstudies by Sano et al.,23 who found an average necrotic areaof 36% in animals undergoing TRAM flap procedures.

These techniques were applied with the purpose of eval-uating whether the stimulation of only a single spot on theTRAM flap right caudal epigastric artery (as in groups 2, 4,and 5 here) would be sufficient to improve viability, andwhether the application of LLLT to 24 sites distributed overand around the flap (as in groups 3, 6, and 7 here) would beenough to stimulate angiogenesis of the flap and its sur-rounding area, thus leading to an increase in TRAM flap vi-ability.

Another determining factor for the application was the en-ergy doses used. Reports in the literature disagree about thisparameter, with doses ranging from 0.082 J/cm2 (in Smithet al.27) to 185 J/cm2 (in Kubota28). In the present study weused doses of 3, 6, 72, and 144 J/cm2 to ascertain the effectof total dose, as well as the effect of a single versus manypoints.

PINFILDI ET AL.340

TABLE 2. SPEARMAN CORRELATION COEFFICIENT RELATED TO

PERCENTAGES OF NECROSIS AND VALUES OF MAST CELLS

Analyzed variable r Value Probability

Necrosis � mast cells A �0.166 0.193Necrosis � mast cells B �0.013 0.920

Significance is set at p � 0.05.

60

50

40

% 30

20

10

0

G1 G2 G3 G4

*

*G5 G6 G7

FIG. 3. Averages of tissue necrosis percentages in the sevenstudy groups.

In the present study, the energy levels used were 0.18, 4.32,0.36, and 8.64 J, which are smaller values than those used byothers. This is a relevant factor to consider, as in the studyby Kubota,28 who used a total dose of 185 J/cm2, but at anenergy level of 1 J, which was less that the levels we used,and those researchers used a dose of 144 J/cm2 and total en-ergy of 8.64 J, which was concentrated on just one applica-tion point. Other studies such as the one by Amir et al.,13

who used energy rates of 1.8 J and 3.6 J and found signifi-cant results, but in a different kind of cutaneous flap.

In the present study, the same energy rates were deliveredin different ways to the tissue, namely on just 1 spot in somecases, and in other distributed over 24 spots in and aroundthe TRAM flap, to assess whether distributing the energymore widely would be better than its application to just onesite.

For analyzing mast cells, toluidine blue dye was used be-cause it is specific for marking mast cells, which become darkblue and are easily seen for morphometry.19

Another mechanism evaluated in this study aside fromblood flow increase, is mast cell exocytosis, which favors his-tamine delivery, improving vasodilation.29

We found increased amounts of mast cells in the groupsirradiated with energy levels of 6 and 144 J/cm2; however,the techniques used to apply the energy were different be-tween the groups. Group 5 was irradiated on only one siteover the pedicle, with total energy of 0.36 J (6 J/cm2), whichshowed an average increase of 1.7. Group 6 was irradiatedon 24 sites inside and outside the TRAM flap, with a totalenergy of 8.64 J (6 J/cm2) which showed an average increaseof 1.4, which differed from the findings in group 7 whichwas irradiated similarly to group 5, with just a single site,and it showed an average increase of 1.7, but with a total en-ergy dose of 8.64 J (144 J/cm2).

The increase in the amount of mast cells in the presentstudy is in line with the findings of El Sayed and Dyson,19

who studied mast cell increase and degranulation with laserirradiation of rat skin wounds (ulcers). In spite of using aninfrared laser at 820 nm, the authors found a significant in-crease in mast cells compared to the control group.

Vasheghani et al.20 also studied the effect of LLLT with avisible red laser on the increase and degranulation of mastcells. The laser used had a wavelength of 632.8 nm (He-Ne),

and in the present study we used a diode laser of 670 nm(InGaAlP).

The results found by Vasheghani et al.20 were obtained inrats that had second-degree burns, and the authors saw asignificant increase in mast cells, mainly in their degranula-tion, in animals undergoing laser therapy at a level of 2.4J/cm2. When comparing energy levels, we can see that thelevels we used in the present study, 3 J/cm2 with just oneapplication site or with 24 sites, did not show any significantincrease compared to higher flows of 6 J/cm2.

According to the results we found, we can see that the ef-fects on mast cells depend on the energy level used, and thusare dose-dependent. Higher flows with only a single site ofapplication to the pedicle of 6 and 144 J/cm2 showed betterresults than those seen in the other experimental groups.

An increase in the amount of mast cells with laser irradi-ation with the 670-nm wavelength has also been confirmedthe findings by Sawasaki et al.,29 who studied LLLT with thesame type of laser and a flow of 8 J/cm2 and total energy

LOWER-LEVEL LASER THERAPY ON MAST CELLS 341

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0G1 G2 G3 G4

*

G5 G6 G7

FIG. 5. (A) Skin sample stained with toluidine blue viewedat 200� magnification. Mast cells after mast cell irradiationat a dose of 144 J/cm2. (B) Similar image at 400� magnifi-cation. Shows degranulated mast cells.

FIG. 4. Averages of mast cell values of skin samples A inthe seven study groups. *, group with the most mast cell av-erage.

B

A

dose of 12 J on oral mucosa in humans. According to the re-sults of both the present study and those of Sawasaki et al.,29

mast cells appear to be sensitive to higher energy levels, asseen with the level of 8.64 J in group 7.

Samples B (viable tissue) which did not receive laser ther-apy, had an increased amount of mast cells, with averagesof 1.3 and 1.4 for groups 3 and 5 respectively, significant re-sults when compared to those of control samples B. Group2 showed a small number of mast cells (0.9 and 0.8) for bothsamples A and B when compared to all other groups, butwithout significant differences compared to the controlgroup, although they showed statistically significant differ-ences compared to groups 3, 5, and 7.

No physiologic answers were found for the finding of mastcell reductions in group 2, which was irradiated with a flowof 3 J/cm2 (0.18J) on only a single site. In terms of tissue ne-crosis, group 2 was the only one showing a higher percent-age of necrosis, with 22.95%. One possible explanation maybe related to the low energy delivered to the tissue, since thegroups showing a significant increase in mast cells were ir-radiated with energy doses of up to 144 J/cm2 (8.64 J).

These findings show that mast cells are sensitive to dif-ferent levels of LLLT, and future studies can be designed tofind the ideal dose to stimulate mast cell degranulation. An-other important perspective would be to study the amountof VEGF and blood vessel formation, to better clarify bothangiogenesis and blood flow increases associated with mastcell increase.

Conclusion

Low-level laser therapy with at 670 nm was effective atreducing the necrotic area in TRAM flaps. In terms of mastcell changes, group 5 (6 J/cm2 at 1 point) and group 7 (144J/cm2 at 1 point) had increases that were greater than thoseseen in the other groups. The technique of applying energyto a single spot on the TRAM pedicle was shown to be bet-ter than the distribution of sites around the flap perimeter,showing that the stimulation of vessel growth responsiblefor flap perfusion is important in increasing the amount ofmast cells, and to reduce flap necrosis.

Acknowledgments

The authors gratefully acknowledge funding from theCNPq (Conselho Nacional de Desenvolvimento Cientéfico eTecnológico) and CAPES (Coordenação de Aperfeiçoamentode Pessoal de Nível Superior).

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Address reprint requests to:Dr. Carlos Eduardo Pinfildi, P.T., Ph.D.

UNIFESP, Plastic Surgery DivisionSurgery Division

Napoleão de Barros St. 715, 4th Floor04024-900 São Paulo, SP, Brazil

E-mail: cepinfildi@hotmail.com

LOWER-LEVEL LASER THERAPY ON MAST CELLS 343