Wound Repair and Regeneration

Effect of low-frequency transcutaneous electrical nerve stimulation (TENS) on the viability of ischemic skin flaps in the rat: An amplitude study Richard Eloin Liebano, PT, PhD1,2; Luiz Eduardo Felipe Abla, MD, PhD1; Lydia Masako Ferreira, MD, PhD1 1. Plastic Surgery Division, Surgery Department, Federal University of Sa˜o Paulo, Sa˜o Paulo School of Medicine, Sa˜o Paulo, Brazil, and 2. Department of Physical Therapy, University of the City of Sa˜o Paulo, Sa˜o Paulo, Brazil

Reprint requests: Richard Eloin Liebano, Rua Napolea˜o de Barros, 715–41 andar, Disciplina de Cirurgia Pla´stica CEP, 04024-002, Vl. Clementino, Sa˜o Paulo, Brazil. Tel: 55 11 5576 4118; Fax: 55 11 5571 6579; Email: [email protected] Manuscript received: January 27, 2005 Accepted in final form: September 17, 2007 DOI:10.1111/j.1524-475X.2007.00332.x

ABSTRACT The purpose of this study was to determine the effect of low-frequency (2 Hz) transcutaneous electrical nerve stimulation (TENS) on the viability of ischemic skin flaps in the rat. Seventy-five EPM1-Wistar rats were used. The random skin flap measuring 104 cm was raised and a plastic barrier was placed between the flap and its bed. After the surgical procedure, animals in all groups were kept anesthetized for 1 hour, with electrodes placed at the base of the flap, and received the assigned treatment. The animals were randomized into five groups (G1–G5) and each group was subjected to the following procedures, which were repeated on the 2 subsequent days: G1—sham stimulation (control); G2—transcutaneous electrical nerve stimulation, TENS (f52 Hz, I55 mA), G3—TENS (f52 Hz, I510 mA), G4—TENS (f52 Hz, I515 mA), G5—TENS (f52 Hz, I520 mA). Seven days after treatment, the percentage of flap necrosis was determined. For each group, the mean  SEM percentage of flap necrosis was as follows: G1 group (control), 43.88  2.02%; G2 group, 39.20  3.17%; G3 group, 38.57  4.08%; G4 group, 32.14  2.89%; and G5 group, 44.13  2.98%. The G4 group had the smallest necrotic area compared with the control group. The lowfrequency TENS treatment was effective in improving the viability of ischemic skin flap.

Skin flaps are frequently used in all areas of plastic surgery especially in reconstructive surgery.1 Ischemic complications are a major source of concern among plastic surgeons who perform flap surgery, because the presence of tissue necrosis in a large number of cases leads to the treatment failure.2,3 Thus, a considerable amount of research has been done to find ways to improve blood flow to the flap reducing ischemic conditions and preventing necrosis of skin flaps.3,4 There are many studies in the literature describing the use of several drugs, such as vasodilators, calcium channel blockers, prostaglandin inhibitors, anticoagulants, antiadrenergics, and antioxidants.3–5 Unfortunately, many of these drugs may lead to undesirable side effects that limit their use in clinical practice.3,6,7 In consequence, new fields of research investigating the effects of nonpharmacological agents, such as acupuncture8 and electroacupuncture,6 pulsed electromagnetic energy,9 low-level lasers,10 low-frequency polarized electrical currents,7,11–14 and nonpolarized currents6,15–18 have emerged. Among these resources, transcutaneous electrical nerve stimulation (TENS) has attracted attention due to its low cost and ease of use. It is being studied as a possible method to improve the viability of ischemic skin flaps.6,7,12,15–18 However, there is a marked variation in the electrical stimulation parameters reported in the literature, especially regarding pulse frequency and current intensity. Kjartansson et al.7 used frequencies of 2 and 80 Hz and intensities c 2008 by the Wound Healing Society Wound Rep Reg (2008) 16 65–69

of 5 and 20 mA in female rat musculocutaneous flaps, and the authors concluded that the use of high-intensity current (20 mA) was the most influential factor in increasing the viability of skin flaps. Niina et al.6 investigated the effects of electroacupuncture and also attempted to reproduce the TENS results of Kjartansson et al.,7 applying both low- (2 Hz) and high-frequency (80 Hz) pulses at intensities of 2, 10, and 20 mA to musculocutaneous flaps in the rat. Their results suggest that high-frequency (80 Hz) pulses have greater influence than current intensity in promoting blood flow and increasing flap viability. However, more recent studies have reported that lowfrequencies (1–5 Hz) are more effective in increasing blood flow.19–29 Based on these findings, the present study was therefore designed to evaluate the effect of low-frequency TENS at different pulse amplitudes on the viability of ischemic skin flap in the rat.

MATERIALS AND METHODS Animals

Seventy-five adult male EPM1-Wistar rats weighing 230– 358 g were used in this study. There was no statistically significant difference in weight among the animals. The animals were housed in individual cages in a temperature65

Effect of low-frequency TENS on ischemic skin flaps

TENS device

The pulse generator used in this experiment was an Orion Tenss (Orion Aparelhos para Fisioterapia LTDA; serial number 00849, Sa˜o Paulo, Brazil). The stimulator generated symmetrical biphasic square wave pulses. The following supplies were also used in this study: one cable, two carbon-impregnated silicon rubber electrodes (4.2 1.5 cm), gel and adhesive tape to hold the electrodes in place. The output of the TENS unit was checked with a digital oscilloscope at the beginning and end of the experiments in the Electrical Engineering Laboratory at the Federal University of Sa˜o Paulo—UNIFESP-EPM. The TENS unit was connected to the oscilloscope and turned on. The frequency of stimulation was set at 2 Hz, the amplitude at 5 mA, and pulse duration at 200 ms. The oscilloscope showed that the output frequency, amplitude and pulse duration were correct. Next, the same process was repeated, but at this time the amplitude was set at 10, 15, and 20 mA, successively. In the case of differences between the oscilloscope readings and the TENS unit settings, the device would be calibrated, but this was not necessary. Operative technique

All animals were anesthetized with tiletamine hydrochloride and zolazepam hydrochloride (50 mg/kg, intraperitoneally) during surgery and TENS applications. Following anesthesia, all rats were placed on a flat surface with legs extended and their backs were shaved. The cranially based dorsal random skin flap, measuring 104 cm, was raised from the deep fascia, including the superficial fascia, panniculus carnosus, subcutaneous tissue, and skin. After flap elevation, a plastic barrier (polyester/polyethylene), with the same dimensions (104 cm) was placed between the skin flap and its bed. The flap was then sutured back in place with simple 4-0 nylon sutures.4,30

200 ms for all treatment groups and the electrodes were removed after each TENS application. Method to estimate the percentage of necrotic area in the distal portion of the flap

The percentage of skin flap necrosis was measured on the seventh postoperative day, using the paper template method described by Sasaki and Pang,31 by an observer who was blind to the treatment condition. Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA), followed by post hoc Dunnett’s multiple comparisons test. The significance level was fixed at 0.05 (p < 0.05). Statically significant values are indicated by an asterisk (n) and nonsignificant values are denoted by NS. Data are presented as mean  standard error of the mean (SEM).

RESULTS For each group, the mean  SEM percentage of flap necrosis was as follows: G1 group (control), 43.88  2.02%; G2 group, 39.20  3.17%; G3 group, 38.57  4.08%; G4 group, 32.14  2.89%; and G5 group, 44.13  2.98%. Only the G4 group had a significantly lower percentage of flap necrosis than control group (p50.032)n. There were no statistically significant differences in necrotic values between groups G2, G3, and G5 and control group (p > 0.05) (NS). Mean percentage of flap necrosis and SEM for all groups are shown in Figure 1.

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G1 = Control

50

G2 = 2 Hz, 5 mA G3 = 2 Hz, 10 mA

40

G4 = 2 Hz, 15 mA 30

G5 = 2 Hz, 20 mA

20 10

TENS procedure

After the surgical procedure, the rats (n575) were randomized into five groups (G1–G5) and kept anesthetized for 1 hour. All animals had electrodes placed on the base of the flap separated by 1.5 cm, and received the assigned treatment, as follows: G1 (n515)—received sham stimulation (control group); G2 (n515)—received TENS (symmetrical biphasic square pulses, f52 Hz, I55 mA); G3 (n515)—received TENS (symmetrical biphasic square pulses, f52 Hz, I510 mA); G4 (n515)—received TENS (symmetrical biphasic square pulses, f52 Hz, I515 mA); and G5 (n515)—received TENS (symmetrical biphasic square pulses, f52 Hz, I520 mA). This procedure was repeated on the 2 subsequent days, with the animals anesthetized according to the same schedule and for the same period of time (1 hour).30 The pulse duration was set at

*

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Necrosis (%)

controlled environment (24  1 1C), on a 12 : 12 hour light– dark cycle (lights on at 7:00 am), and fed standard rat chow and water ad libitum. This research was approved by the Research Ethics Committee (CEP) at the UNIFESP/EPM, Sa˜o Paulo, Brazil, under the process number CEP 0380/03.

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Groups

Figure 1. Dot plot representing percentage values of flap necrosis. Dots represent the percentage of necrosis for each animal, and vertical lines represent mean and SEM in groups. ANOVA—Dunnett ControlG2 ControlG3 ControlG4 ControlG5 n

(p50.564) NS (p50.666) NS (p50.032)n (p50.999) NS

Significantly different as compared with control (p > 0.05). c 2008 by the Wound Healing Society Wound Rep Reg (2008) 16 65–69

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DISCUSSION Skin flap necrosis can be attributed to both extrinsic and intrinsic factors. Extrinsic factors include systemic (infection, arteriosclerosis, hypotension, or malnutrition) and local (compression, tension, or thrombosis at the anastomosis) alterations, while inadequate blood flow is the only intrinsic factor.2 Therefore, modalities that increase arterial inflow to the skin flap must be studied.2,18 The TENS is a well-known, low cost and easy-to-use therapeutic source to control acute and chronic pain.32 Besides the analgesic effect, some researches demonstrated that TENS application alters the temperature and cutaneous blood flow19,20,23,33,34 enlarging the range of indications for the use of TENS in the treatment of chronic ulcers22,25,35,36 and ischemic skin flaps.6,7,18 The present study showed that TENS treatments may increase the viability of skin flap in a rat model. In this study, the frequency of 2 Hz was used based on recently published studies pointing to the superiority of lower frequencies (1–5 Hz) over the higher frequencies (75–150 Hz) in increasing cutaneous blood flow.25–29 Stimuli were delivered at different amplitudes to five groups of rats and G4, which correspond to a current amplitude of 15 mA, was the only group to show significant decrease in skin flaps necrosis. Similar results are found in previous studies using the same experimental protocol but rather applying high-frequency (80 Hz) TENS.37 Kjartansson et al.7 comparing low- (2 Hz) and high-frequency (80 Hz) TENS at amplitudes of 5 and 20 mA, concluded that the use of highintensity electrical stimulation (20 mA) was the most influential factor in increasing blood flow and flap survival. In the present study, current amplitude of 20 mA did not prove effective in increasing the viability of skin flaps. This contradiction may be explained by differences between the two skin flap models, stimulation pulse parameters, or electrode size.37 The influence of current amplitude on the improvement of the viability of skin flaps is not generally accepted in the literature. Niina et al.,6 using TENS at low- (2 Hz) and high(80 Hz) frequencies with current amplitudes of 2, 10, and 20 mA, concluded that the use of high frequency is the most influential factor independent of the amplitude. Some caution should be exercised in analyzing these results because only a small number of animals were used and this result is not supported by other studies.7,12,14,15,17,18,37 In the present study, all TENS experimental groups except G5 group, showed a lower mean percentage of flap necrosis compared with the control group. However, there was a statistical difference only between the G4 group and the control group. This may be due to a wide range of flap necrosis usually observed in this type of model. This variation is reported by other studies using the same model of skin flap4,18,30,37,38 and has not been satisfactorily explained in the literature. Similar findings can be found in other studies.7,15–17,39 When studying TENS effects on the viability of ischemic skin flaps, Kjartansson et al.7,39 reported an increase in blood flow and survival rates in rats and patients.15–17 However, in another study, no significant increase in blood flow was observed when applying TENS to three ischemic flaps in humans.40 One possible explanation may be a variation in the degree of ischemia in the flaps.40 Although a random-pattern skin flap in rats c 2008 by the Wound Healing Society Wound Rep Reg (2008) 16 65–69

Effect of low-frequency TENS on ischemic skin flaps

was used in the present study, minor variations in the degree of ischemia may be the responsible for TENS results and large necrosis variation. It is known that approximately 30% of patients with chronic pain do not respond to TENS.41 Like these patients, the ischemic skin flaps may also fail to respond to TENS. The exact explanation for this observation is still unclear. Individual biological variations may influence how skin flaps react to TENS, whether favorably or not. In this study, the electrical stimulation was applied in transcutaneous mode because it is a noninvasive procedure and frequently used by other researchers.7,12,13,15–18,37,38 Also, a previous study showed that electrical stimulation through surface electrodes was effective in increasing the viability of musculocutaneous flaps, unlike the stimulation applied with needle electrodes (electroacupuncture).6 It is difficult to compare results of different studies using TENS in flap survival as there is a lack of specific information about the experimental protocol. In some published works there are no data about electrode size,7 distance between electrodes6,7,12 and pulse duration.13 In a previously published study,30 we proposed an experimental model for the study of TENS effects on skin flaps in the rat. It is still unclear what mechanism is involved in the increase in cutaneous blood flow by TENS; however, some hypotheses have been proposed in the literature. One of them argues that TENS, through the stimulation of largediameter mechanosensitive fibers, may cause inhibition of the sympathetic vasoconstrictor neurons, resulting in cutaneous vasodilation.7,33,42,43 According to a more favored hypothesis, TENS application would cause the liberation of some vasodilating neuropeptides such as the substance P (SP), calcitonin gene-related peptide (CGRP), and vasoactive intestinal polypeptide (VIP).7,12,14,19 The hypotheses of Nolan et al.21 and Cramp et al.29 state that an increase in the blood flow may be justified by the muscle pump or by an increase in the metabolic demand of the stimulated muscles when the stimulation is performed at motor level. A new proposed method to increase flap viability is the administration of opioids.44 It is known that electrical stimulation, especially in low frequencies, can induce the release of opioids.45–47 This could be a new hypothesis to explain the effects of TENS on flap viability. There are new ideas to pursue in the search for new physical agents that would minimize post-operative complications of skin flaps. New studies on the mechanism of action of electric currents in ischemic flaps, controlled clinical studies, as well as the use of other physical agents can contribute to a better understanding to an important aspect of reconstructive surgery.

ACKNOWLEDGMENT This work was financially supported by CAPES, a Brazilian funding agency.

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