Effect of electrical stimulation on survival of skin flaps in pigs.

There is increasing evidence that electrical stimulation affects tissue repair. Treatment with electrical current can improve osteogenesis in bone healing [1-3] and may accelerate the healing of skin wounds, [4,5] including ischemic ulcers. [6-8] Ischemic skin ulcers demonstrate chronic epidermal and dermal erosion and significant local hypoxia. Tissue damage occurs secondary to both ischemia and other local processes.

Skin flaps and grafts are essential to the successful functional and esthetic reconstruction of defects of congenital, traumatic, or malignant origin. The major problem related to the use of skin flaps concerns necrosis of a portion of the flap during its preparation and utilization. Prolonged vascular occlusion (ischemia), followed by reperfusion, in island skin flaps causes complete tissue necrosis. [9] The viability of skin flaps subjected to ischemia and reperfusion can be enhanced by treatment with either superoxide dismutase (a scavenger of the superoxide-free radicals),[10] allopurinol (an inhibitor of superoxide production from xanthine oxidase), [11] mannitol and dimethyl sulfoxide (hydroxyl radical scavengers), [12] or deferoxamine (iron chelator-inhibiting hydroxyl radical production). [13] These findings suggest that oxygen-derived free radicals are important mediators of the tissue injury that occurs during reperfusion following ischemia.

Acute island skin flaps and pedicle flaps exhibit an increasing gradient of ischemia and consequent tissue necrosis toward their distal margin. The concept of an initial hyperadrenergic state in such skin flaps has been proposed. [14] An accumulation of the neurotransmitter, norepinephrine, follows transection of the sympathetic nerves, and the resultant vasoconstriction induces a temporary, but complete, capillary occlusion. Subsequently, the capillaries may become dilated and allow reperfusion after the neurotransmitter has been cleared from the tissues. This sequence of events has been proposed to take place in the transition zone between adequately perfused proximal tissue and inadequately perfused distal ischemic areas. Because this mechanism involves ischemia followed by reperfusion, we postulate that necrosis in the transition zone is mediated by oxygen-free radicals and can he ameliorated by treatment with free-radical scavengers. [15]

Negative electrical current blocks sympathetic vasoconstriction,[16] and initial treatment of skin flaps with negative-electrode stimulation may prevent severe ischemia, thus preventing the tissue injury that occurs during reperfusion following ischemia. Theoretically, positive-electrode stimulation can neutralize superoxide anions by the flow of protons, and treatment with positive-electrode stimulation may prevent free-radical-mediated tissue damage. We hypothesize, therefore, that the injury might be ameliorated by treatment with combined negative-positive electrical current. The present study was performed to test our hypothesis that electrotherapy would demonstrate a beneficial effect on the survival rate of skin flaps.

Method

Twelve Yorkshire pigs (Sus scrofa), weighing 15 to 20 kg, were used for the experiments. After the animals were sedated with an intramuscular injection of ketamine (20.0 mg/kg), acepromazine maleate (1.0 mg/kg), and atropine (0.2 mg/kg), they were intubated with an endotracheal tube and mechanically ventilated. Pentobarbital 20-30 mg/kg) was given intravenously to maintain a satisfactory level of anesthesia during surgery. Four bipedicle skin flaps measuring 4 x 20 cm were created on each animal, two flaps on each flank. The skin flaps were created by means of parallel incisions, with undermining in the superficial fascial plane beneath the panniculus carnosus, preserving the dorsal and ventral pedicles. The flaps were sutured into position in their donor sites. The ventral pedicles were situated 4 cm from the nipple line, and the dorsal pedicles were situated 3 to 4 cm from the dorsal midline. The flaps were separated from each other by a distance of 6 cm (Fig. 1).

The animals were placed in a body sling during electrotherapy. The most ischemic area of the skin flaps was identified on the basis of tissue glucose and lactate as being 9 to 13 cm from the ventral pedicle (Fig. 2). The 0.1-mg tissue segments were treated with 20 [micro]L of 0.01 N HCI for 10 minutes at 60 degrees C. Glucose and lactate were assayed by fluorometric enzymatic methods. [17] The ischemic areas of the flaps were treated with pulsed electrical current using the Staodyn Vara/ Pulse stimulator. The ischemic areas were covered with saline-soaked gauze, and the electrode pads were placed on the gauze and secured in place. The water-saturated dispersive pads were placed on the animal's buttocks (Fig. 1) and secured with Velcro belts. The intensity of the electrical current was 35 mA, and the frequency was 128 Hz. Control animals received either a sham treatment (n = 2) or no treatment (n = 3). The experimental animals (n = 7) were stimulated with the negative electrode for 30 minutes postsurgery. Three experimental animals received a second 30-minute application of negative-electrode stimulation 4 hours after the first treatment. The initial treatment was administered 0.5 to 2.5 hours after the operation. During the next two days, the skin flaps were stimulated with the negative electrode for 30 minutes twice daily. The skin flaps were stimulated with the positive electrode during days 4 to 6, and again with the negative electrode during days 7 to 9. The treatment schedule was based on an ongoing clinical trial involving patients (Katherine Miller, Staodynamics, Inc; personal communication; December 1986) and on the published protocol. [6] The length of viable flap and the extent of skin necrosis were measured (in centimeters) on postoperative day 21. Skin necrosis was assessed by the gross appearance of the development of dark and hardened tissue. The mean proportion of necrosis for each group was expressed as a percentage of total flap area.

Results

In the control groups, no significant difference was found between the sham-treated and untreated animals; an average of 28% of each flap became necrotic. The area of necrosis in the electrically stimulated animals was significantly reduced from 28.0% to an average of 13.2% (p < .001, Student's t test) (Tab. 1). Skin flaps exhibiting either complete survival or tissue necrosis of less than 10% were considered improved. The skin-flap survival rate is depicted in Table 2. Fifty percent (14/28) of the flaps in the treatment group demonstrated significant improvement (p < 001, Fisher Exact Probability test [18]). The majority of the improved flaps (10/14) exhibited no sign of tissue necrosis. Experimental animals receiving one negative-electrode treatment on the first day demonstrated a lower skin-flap survival rate than animals receiving two negative-electrode treatments.

Discussion

Chemical reactions elicited by electrodes may be the primary stimulus for tissue response. The external application of direct current may produce a flow of electrons and protons through tissues. The reaction at the negative electrode (cathode) has been shown to consume dissolved oxygen with the production of hydroxyl ions (2H[.sub.2]0 + 0[.sub.2] + 4e- > 40H-), resulting in a decrease in tissue oxygen tension. [19,21] The maintenance of a low partial pressure of oxygen (PO[.sub.2]) can be one of the principal mechanisms by which cells are protected from oxygen toxicity and lipid peroxidation in vivo. Sources of superoxide production are responsive to PO[.sub.2], and the production of oxygen-derived free radicals is decreased in the presence of low tissue PO[.sub.2]. [21]

Theoretically, the positive electrode (anode) could scavenge the superoxide radicals (0[.sub.2]-) by the flow of protons (0[.sub.2]- + 0[.sub.2]- + H[.sup.+] > 0[.sub.2] + H[.sub.2]O[.sub.2]). Thus, treatment with positive-electrode stimulation in the later stages of tissue repair may prevent tissue injury by scavenging superoxide radicals. In pilot experiments, electrodes were placed in a reaction mixture of xanthine, xanthine oxidase, and nitroblue tetrazolium (NBT). The reaction of xanthine oxidase produces superoxide radicals, which reduce NBT (pale yellow color) to formazan (purple color); this reduction is inhibited by superoxide dismutase. [22] In this system, no color (formazan) was formed in the vicinity of the anode, indicating neutralization or spontaneous dismutation of superoxide anions by movement of protons.

The time interval between the skin-flap operation and the initial treatment may be a critical factor. In hyperbaric oxygen therapy, the initial treatment should be administered within four hours of the operation to produce beneficial effects. [23] in the present study, the initial treatment was administered within 2.5 hours postoperatively. Animals receiving a single treatment on the first day demonstrated significantly lower skin-flap survival rates than those receiving two treatments: 19% (3/16) and 92% (11/ 12), respectively. The present data suggest that the beneficial effect of electrotherapy may be related to the early response to stimulation. Further investigations with larger groups of animals should be performed to substantiate the importance of the initiation time of treatment and dosage.

Conclusion

It is evident from previous studies that oxygen-derived free radicals play an important role in ischemiare-perfusion injury in skin flaps. [1-13] The present investigation demonstrates that pulsed electrical stimulation can improve the survival of skin flaps. initial treatment with the negative electrode may prevent severe ischemia by blocking sympathetic vasoconstriction [16] and negate the ischemia-reperfusion event occurring in the transition zone in the skin flap. Alternate treatment with positive-electrode stimulation may produce a free-radical-scavenging effect by neutralization or dismutation of superoxide anions.

References

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(Tables and other figures omitted)