Two hours of electrotherapy was followed by four hours of rest. This 2:4-hour treatment regimen was repeated three times daily, seven days a week, until wound ...
Electrical Stimulation Effects on Cutaneous Wound Healing in Rabbits A Follow-up Study MARYBETH BROWN, MARY KATE McDONNELL, and DAVID N. MENTON The purpose of this study was to determine the effects of high voltage monophasic pulsed electrical stimulation on wound healing using positive polarity. Forty-four rabbits were assigned to experimental or control groups and followed for four or seven days. We classified the groups as Exp4, Con4, Exp7, and Con7, respectively. Each animal was anesthetized, and a full-thickness incision, 3.5-cm long, was made on its back. After 24 hours, the Exp4 and Exp7 rabbits received high voltage electrical stimulation for two hours twice daily. Wound closure for the Exp4 rabbits (50%) was significantly less than that of the Con4 rabbits (78%). After seven days, however, the Exp7 and Con7 rabbits had similar wound-closure values (80% and 82%, respectively). Tensile-strength values for the control and experimental animals were comparable at both time periods. Histologic examination of the wounds suggested a more rapid rate of epithelization between the Exp4 and Exp7 rabbits compared with the Con4 and Con7 rabbits. The results of this study are inconclusive, but may indicate that positive-polarity stimulation enhanced wound closure between four and seven days of treatment. Key words: Electric stimulation, Histological technics, Tensile strength, Wound healing.
Some evidence exists that cutaneous wound healing can be enhanced if direct current (DC) electrical stimulation is used.1"6 Several investigators have used negative polarity low voltage stimulation to enhance wound healing.1-3 Assimacopoulos found that complete wound closure occurred earlier in two rabbits stimulated continuously with 100-A, 0.8- to 1.1-V, negative-polarity DC than in two rabbits that received no stimulation.1 Complete wound closure in the treated animals occurred after 18 and 19 days, respectively, whereas the wounds of the two untreated rabbits were considered completely healed after 25 and 26 days, respectively. Wu et al, however, found no difference in the tensile strength of wounds treated with positive or negative current.2 They made bilateral incisions on the abdomen of 26 rabbits and sutured them with stainless steel wires. A negative current was then passed through one incision, and a positive current was passed through the other incision. Current intensity, delivered from a 1.5-V battery, ranged from 40 to 400 µA. Whether the tensile-strength values for control rabbits were greater or lesser than tensile-strength values from treated animals was not mentioned.2 Alvarez and co-investigators reported enhanced wound healing in pigs treated with positive-polarity DC.4 Alvarez et al made 0.3-mm deep wounds on the dorsal skin of the pigs M. Brown, PhD, is Assistant Professor, Program in Physical Therapy, Washington University Medical School, 660 S Euclid Ave, PO Box 8083, St. Louis, MO 63110 (USA). M. McDonnell, MHS, is Physical Therapy Supervisor, Department of Physical Therapy, Irene Walter Johnson Rehabilitation Institute, 509 S Euclid Ave, St. Louis, MO 63110. D. Menton, PhD, is Associate Professor, Department of Anatomy and Neurobiology, Washington University Medical School. This article was submitted February 10, 1987; was with the authors for revision 14 weeks; and was accepted August 13, 1987. Potential Conflict of Interest; 4.
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and treated the wounds continuously, for up to seven days, with 50 to 300 µA of DC. Treated pigs healed faster and showed a significant increase in collagen synthetic activity when compared with untreated pigs.4 Other investigators found improved healing when polarity was changed during the course of treatment.5,6 Using low intensity DC ranging from 200 to 1,000 µA, Gault and Gatens reported twice the rate of healing of 100 ischemic skin ulcers in human patients.5 The negative electrode was applied to the ulcer the first three days, and a positive electrode was used thereafter. Two hours of electrotherapy was followed by four hours of rest. This 2:4-hour treatment regimen was repeated three times daily, seven days a week, until wound healing occurred. Wound healing appeared to be enhanced in each of these studies using DC, regardless of polarity.1-6 Most clinics, however, currently appear to favor the use of high voltage stimulation (HVS) over DC stimulation. The use of HVS for wound healing has not been well explored. In a pilot study by Feedar and Kloth, five patients with decubitus ulcers were treated successfully with pulsed HVS.7 The polarity of the active electrode was negative for the first three days and positive thereafter. The voltage used was that amount required to cause a barely visible contraction; current frequency was 150 Hz. Results of a previous study by Brown and Gogia using negative-polarity HVS for wound healing were conflicting.8 Six rabbits treated with HVS showed, after four days, a trend toward higher tensile-strength values than nine untreated rabbits. After seven days of HVS treatment, however, 10 control rabbits had significantly greater tensile strength values than 9 treated rabbits. Thus, the negative-polarity HVS appeared to hamper the healing process in the treated animals between four and seven days postoperatively. 955
This study was conducted to further delineate the role of HVS in wound healing and to determine the effects of positive polarity on the wound-healing process. Specifically, the study was designed to 1) assess the extent of wound closure in rabbits treated with HVS and in untreated rabbits, 2) measure the tensile strength of wounds of treated and untreated rabbits, and 3) examine histologically wounds of treated and untreated rabbits. METHOD Animals Thirty-two female adult (4-6 months of age) New Zealand rabbits, weighing between 2 and 2.5 kg, were purchased from a local breeding farm* and housed, one per standard cage, in a vivarium. When received, the rabbits were randomly assigned a number that was printed on the ear with indelible ink. Animals remained in their cages, except during treatment set-up, where food and water were available ad libitum. Temperature (22°C) and the ratio of daylight hours to nondaylight hours (12 hours light/12 hours dark) were kept constant. Rabbits were divided into two experimental groups and two control groups. One experimental group (n = 10) and one control group (n = 6) were each followed for four days (Exp4, Con4). The other experimental group (n = 10) and control group (n = 6) were each followed for seven days (Exp7, Con7). Only six animals were placed in each control group because data regarding tensile strength and percentage of wound closure were already available from Brown and Gogia's previous study on 9 control rabbits followed for four days and 10 control rabbits followed for seven days.8 Skin Lesion The process of creating a skin wound was identical to that used in the previous study by Brown and Gogia.8 The dorsal aspect of the thoracic area was shaved and anesthetized, and a full-thickness incision about 3.5 cm in length was made. Skin pieces were then approximated, and Steri-Strips®† were applied to maintain wound closure. Furasin®‡ powder was sprinkled liberally over the wound area, and the animals were bandaged and wrapped with an elastic bandage. Treatment Treatment was administered in the same manner as in the previous study,8 except that polarity was positive. Before treatment, bandages were removed and a 4.5- × 1.2-cm carbon-rubber active electrode§ coated with a salt-free electrode gel" was applied directly over the wound site and taped in place. Several centimeters laterally, the dispersive electrode was applied. Control rabbits had gel-coated electrodes applied to the wound site but were not treated with HVS. Every morning and afternoon, HVS was given for two hours per session to the Exp4 and Exp7 animals. During each two-hour treatment session, the animals were free to move about in * Boswell Bunny Farm, Inc, Rt 1, PO Box 33, Pacific, MO 63069. † 3M, Medical-Surgical Div, Bldg 225-5S, 3M Center, St. Paul, MN 551441000. ‡ Norwich Eaton Pharmaceuticals, Inc, Norwich, NY 13815-0231. § Medtronic, Inc, 7000 Central Ave NE, PO Box 1250, Minneapolis, MN 55440. || Parker Laboratories, Inc, 307 Washington St, Orange, NJ 07050.
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Fig. 1. Skin piece arranged on a frame and clamped in place at top and on both sides. Vice-grip pliers and plastic water bag were attached below, but near the middle of, the wound site (arrow).
their cages. Treatment was given for a total of three days (6 sessions) to Exp4 rabbits and six days (12 sessions) to Exp7 rabbits. The HVS used was a twin-peaked, monophasic waveform (reverse sawtooth). The current was pulsed, with a pulse width of 100 µsec, and delivered at a rate of 80 pulses per second. The stimulating unit# was turned on until barely palpable contractions (threshold current) were felt on each rabbit. Voltages ranged between 30 and 60 V. Rabbits were checked every few minutes, and the current was increased or decreased as necessary. Measurements Wound closure. At the end of the experiment, the animals were anesthetized with ether and killed with a 2-cc intracardiac injection of 2% potassium chloride. We then removed the bandages and used a cloth tape measure to measure the length of the entire wound and the portion of the wound that was closed. Tensile strength. After measuring each wound, we removed an area of skin containing the wound. The procedure for obtaining tensile-strength measurements was described previously.8 We mounted the rectangular skin piece by clamping the top and both sides to a frame. On the lower (undamped) portion of skin, near the middle of the wound site, a pair of vice-grip pliers and a lightweight plastic bag were attached (Fig. 1). Subsequently, water was added to the plastic bag until the wound failed, that is, opened completely. If the wound was not opened completely by the time the bag was full of water, small weights were attached to the bag in 10- to 20-g increments. Tissue Examination Immediately after the tensile-strength portion of the study, skin pieces were trimmed so that only a portion of skin containing the wound remained. The skin pieces were flattened and fixed in 10% neutral buffered formalin for several days. After fixation, the tissue was embedded in paraffin, sectioned at 6 µm with a microtome, and stained with hematoxylin and eosin (H&E) for routine morphological # EGS Model 300, Electro-Med Health Industries, Inc, 6240 NE 4th Ct, Miami, FL 33138. PHYSICAL THERAPY
RESEARCH
analysis and with Verhoeff-van Gieson's stain (VVG) for differentiation of skin layers and collagen.9 Sections were cut perpendicular to the wound site permitting visualization of the epidermis and the dermis in the wound area (Fig. 2). Tissue sections were obtained from the middle of the wound and from an area about 1 cm from the edge of the wound. Slides from each rabbit were examined with the aid of a light microscope. Three criteria were examined for all animals: 1) the amount of epithelization that had taken place four and seven days postinjury; 2) whether wound closure (or lack of wound closure) occurred at the level of the epidermis or dermis, or both; and 3) the relative amount and fiber architecture of collagen present in the dermis, proximal to the wound site. During the healing process, the stratum basale (active cellproducing layer of the epidermis) proliferates, partially filling and bridging the incision.10,11 The increase in thickness of the proliferating epidermis can be measured using an eyepiece reticle, a component of the microscope. As a relative index of cellular regeneration, the thickness of the epidermis was measured at the wound site. The increase in thickness was expressed as "minimal" if the increase was 2 to 3 x greater than the epidermal thickness found in uninjured skin, "moderate" if the increase was 5 to 6 x greater, and "maximal" if the wound epithelium had increased in thickness 10 x or more. If no change in the epidermis was noted, the lack of change was recorded as "none." Quantity of collagen at the wound site was subjectively designated as "sparse," "moderate," or "dense." Sparse indi-
cated that the amount of collagen present was about one quarter that observed in an unaffected portion of the dermis. Moderate indicated that the amount of collagen present at the wound site (presumably new collagen) was half that observed in an unaffected portion of the dermis, and dense indicated three quarters or more. Orientation and size of the collagen fibers adjacent to the wound site were also noted. Eight Additional Animals
Histologic observations at four and seven days postinjury indicated that the HVS particularly affected the rate of cell division of the stratum basale of the epidermis. Because we were examining wounds that had been pulled apart to obtain tensile-strength measurements, we were never sure whether our observations were valid. Some of the epidermis was possibly damaged or lost during the tensile-strength portion of the study. To further evaluate the effects of HVS on the epidermis, we extended the scope of the study by examining eight more rabbits. The eight additional animals were obtained from the same breeding farm and assigned two each to the experimental and control groups. These animals were treated in precisely the same manner as those mentioned previously with either two hours of HVS administered each morning and afternoon or no HVS treatment. After four or seven days postinjury, these animals were also anesthetized and killed. Wound closure was noted, but in this instance no wound failure values were obtained. Wounds were harvested intact, placed in 10% neu-
Fig. 2. Cross-section of skin from a treated rabbit four days postinjury. The dark band at the top of the photo (E) is the epidermis. The dense material above the epidermis (X) is a scab. The wound is closed only at the level of the epidermis. A distinct gap is visible in the dermis (D) where the scalpel penetrated (arrows). Notice that the epidermis above and adjacent to the wound site is thicker than the epidermis further away, indicating a proliferation of cells in the germ layer of the epidermis at the wound site. (H&E stain, 88 x before reduction.)
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tral buffered formalin, embedded in paraffin, sectioned perpendicular to the wound, and stained with H&E and VVG. The entire wound of these animals (ie, epidermis, scab, dermis, and precisely where the scalpel blade had penetrated) could be visualized.
TABLE 1 Percentage of Wound Closure
RESULTS Groups Exp4 and Con4 Wound closure. Although differences in percentage of wound closure were not statistically significant (p > .05), Con4 rabbits had larger values for wound closure (78%) after four days than did Exp4 animals (50%) (Tab. 1). Eight of the 13 Con4 animals had wounds that were completely closed, whereas 3 of the 9 Exp4 animals had completely closed wounds. Tensile strength. Four days after injury, tensile-strength values for treated and untreated animals were not significantly different. Tensile-strength values for the Con4 animals ranged from 451 to 2,328 g, whereas values for the Exp4 animals ranged from 536 to 2,956 g (Tab. 2). Histological observations. After four days of healing, the wound was closed or partially closed in most animals. Closure usually was accomplished by the epithelium, although in most animals new collagen could be observed at the wound site in the dermis. Typically, fibroblasts were seen in abundance in the dermis, and the epidermis had been generating new cells. The wound-closure process appeared delayed in treated rabbits. Two Exp4 rabbits showed no increase in epidermal thickness, nor had the epidermis begun to bridge the gap created by the scalpel. Four Exp4 animals (41%) had a minimal increase in epidermis thickness at the wound site; three had moderate increases. By contrast, only two Con4 rabbits had a minimal increase in epidermis thickness; the remainder had a moderate increase. 958
Exp4 Con4 Exp7 Con7
9 13 8 14
Percentageb
50.4 77.7 79.9 82.4
39.6 31.1 32.0 32.0
a Number of animals from which wound-closure values could be obtained. Data of 9 Con4 and 10 Con7 animals were obtained in the previous study.8 b Values are not statistically significant.
TABLE 2 Tensile-Strength Values (g) Group
na
Exp4 Con4 Exp7 Con7
9 13 8 14
Tensile Strengthb s
Data Analysis Wound size (length) differed from animal to animal. To normalize data, the percentage of wound closure for each animal was obtained. If a wound was 3.5 cm in length and 2.5 cm of the wound was healed (closed), for example, the percentage of wound closure for that animal was 71%. We used a Student's t test to determine significant (p < .05) differences in percentage of wound closure between Exp4 and Con4 animals and between Exp7 and Con7 animals. The Student's t test was also used to determine significant differences in tensile strength between control and experimental animals.
na
s
Animal Loss One Exp4 animal and two Con4 animals were eliminated from the study because of illness or wound tampering. Consequently, results are presented for 9 Exp4 and 13 Con4 animals. Four of the Con4 animals were from this study; nine were from the previous study.8 Appropriate statistical tests were performed to ensure that data from the two groups of animals could be combined. Two Exp7 and two Con7 animals were excluded from the data analysis. One animal had an infected wound, two animals tampered with their wounds, and one animal was eliminated because of illness. Data, therefore, are presented for 8 Exp7 and 14 Con7 animals. Four of the Con7 animals were obtained from this study; 10 were from the previous study.8
Group
1,668 1,424 2,240 2,665
803 882 711 1,270
a
Number of animals from which tensile-strength values could be obtained. Data of 9 Con4 and 10 Con7 animals were obtained in the previous study.8 b Tensile-strength values are nonsignificant.
Differences in the amount of dermal collagen present at the wound site were less apparent when Con4 and Exp4 rabbits were compared. Eight of the nine Exp4 rabbits had a sparse quantity of collagen present; one rabbit was rated as having a moderate amount of collagen. Two thirds of the Con4 rabbits had a sparse quantity of collagen; one third were rated as having a moderate amount. Fibroblasts and new collagen fibers appeared irregular in orientation in tissues from both groups of rabbits. No differences in wound appearance could be discerned when slides of sections taken from the central portion of the wound were compared with sections taken from the edge of the wound site. Groups Exp7 and Con7 Wound closure. After seven days of HVS treatment, woundclosure values of the Exp7 animals were comparable with those of the control animals. Between four and seven days postinjury, wound-closure values for treated animals increased from 50% to 80%, whereas the values for the control animals increased only from 78% to 82% (Tab. 1). Tensile strength. Values for tensile strength among rabbits treated seven days were not statistically different. Tensilestrength values for the Exp7 and Con7 animals were greater than those for the Exp4 and Con4 animals (Tab. 2). Histological observations. After seven days of treatment, experimental animals showed histologically noticeable improvement. Five of the eight Exp7 animals (62%) had a moderate increase in epidermis thickness; two had minimal increases, and one had a maximal increase. One third of the Con7 animals had a minimal increase in epidermis thickness; the other two thirds showed a moderate increase. Collagen density in the dermis had increased for both Con7 and Exp7 rabbits. Only two Con7 and three Exp7 animals PHYSICAL THERAPY
RESEARCH had a sparse rating. The remaining 12 Con7 animals were rated as having a moderate density of new collagen at the wound site, and the other 5 Exp7 animals had a moderate (n = 3) or dense (n = 2) amount of new collagen present. One observation, not apparent in Exp4 animals, was that fibroblasts and collagen fibers located in the dermis of Exp7 animals tended to be regularly arranged. Rather than being randomly oriented, as was typical in the Con7 rabbits, fibroblasts and collagen fibers immediately beneath the epidermis were aligned parallel to the epidermis in five of the Exp7 rabbits. Results for Eight Additional Animals Observations for these additional animals confirmed the results discussed previously. Epidermal proliferation appeared to be just starting in the two Exp4 rabbits as compared with the two Con4 rabbits. By contrast, the amount of change in the two Exp7 animals was considerable; the increase in epidermal thickness was rated as maximal in both Exp7 rabbits and as moderate in the two Con7 animals (Fig. 3). DISCUSSION The major question of interest, whether positive-polarity HVS treatment enhances wound healing, was not answered by this study. A tendency for delayed healing was observed for animals treated four days postoperatively. The delayedhealing trend in the Exp4 rabbits suggests that positivepolarity HVS, as administered in this study, is contraindicated between one and four days postoperatively. A trend toward delayed wound closure between one and four days postoperatively using positive-polarity HVS was noted in this study. Previous findings include significantly reduced tensile-strength values in wounds treated between four and seven days postoperatively with negative-polarity HVS.8 These two findings suggest 1) that polarity is a major factor contributing to treatment outcome with HVS and 2) that use of positive-polarity HVS between one and four days postoperatively and negative-polarity HVS between four and seven days postoperatively will retard the wound-healing process. A tendency toward improved wound healing was observed when negative-polarity HVS was used between one and four days postoperatively8 and when positive-polarity HVS was used between four and seven days postoperatively. The tendency toward enhanced wound healing, however, must be viewed in light of the potential danger of causing harm with electrical stimulation. Further study to provide substantive evidence of the safety and efficacy of negative and positive HVS is indicated.
Although polarity did not appear to be a factor contributing to enhanced wound healing with DC in previous studies,1"6 polarity of the active electrode was an important factor in this study and in the previous study by Brown and Gogia.8 Evidence for why polarity was a factor related to success with HVS may have been provided by Burr et al who measured electric current potential at the wound site in guinea pigs.12 These investigators made full-thickness incisions on the back of 10 animals and found that for the first three or four days electric potentials were positive but after the fourth day electric potentials at the wound site became negative and remained negative until complete healing occurred.12 This finding may explain the trend toward enhanced wound healing observed in rabbits treated with negative-polarity HVS for four days postoperatively8 and in the rabbits in this study treated with positive-polarity HVS between four and seven days postoperatively. The findings by Burr et al may also explain why animals positively stimulated the first four days postoperatively and negatively stimulated between four and seven days postoperatively exhibited poor wound healing when compared with controls. Although polarity of the active electrode emerged as a major factor contributing to treatment outcome, other factors also affect the healing process.13,14 The results of this study and of the previous study by Gogia and Brown8 were not unequivocal. That is, clear-cut differences between treated and untreated animals were not apparent in either study, even though the rabbits were all about the same age; were seemingly in good health; ate the same food; and were subjected to similar, stringent treatment protocols. Perhaps complex ionic, physical, or electromagnetic phenomena affected the healing process.13,14 Additional study is needed to determine how these factors affect healing and how physical therapists can use physical, ionic, and electromagnetic phenomena to their advantage in treating patients. CONCLUSIONS Wound-closure values for rabbits treated with positivepolarity HVS for four days were 28% less than those for controls. Treatment given under conditions similar to those discussed in this study, therefore, is contraindicated. Positivepolarity HVS appeared to accelerate wound closure between four and seven days of treatment. Histological evidence indicates that wound closure in the treated animals occurred by epithelization. Tensile strength was not affected by the treatment procedure used in this study. Acknowledgment. We acknowledge the invaluable assistance of Electro-Med Health Industries, Inc, who loaned us all of the electrical stimulation equipment used in this study and provided helpful suggestions as well.
REFERENCES 1. Assimacopoulos D: Wound healing promotion by the use of negative electric current. Am Surg 34:423-431,1968 2. Wu KT, Go N, Dennis C, et al: Effects of electric currents and interfacial potentials on wound healing. J Surg Res 7:122-128,1967 3. Wolcott LE„ Wheeler PC, Hardwicke HM, et al: Accelerated healing of skin ulcers by electrotherapy. South Med J 62:795-801,1969 4. Alvarez OM, Mertz PM, Smerbeck RV, et al: The healing of superficial skin wounds is stimulated by external electrical current. J Invest Dermatol 81:144-148,1983 5. Gault WR, Gatens PF Jr: Use of low intensity direct current in management of ischemic skin ulcers. Phys Ther 56:265-269,1976 6. Thurman BF, Christian EL: Response of a serious circulatory lesion to electrical stimulation. Phys Ther 51:1107-1110,1971 7. Feedar JA, Kloth LC: Acceleration of wound healing with high voltage pulsating direct current. Abstract. Phys Ther 65:741,1985
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8. Brown M, Gogia PP: Effects of high voltage stimulation on cutaneous wound healing in rabbits. Phys Ther 67:662-667, 1987 9. Lillie ED, Fullmer HM: Histopathologic Technic and Practical Histochemistry, ed 4. New York, NY, McGraw-Hill Inc, 1977, pp 126-132 10. Bassett CAC, Hermann I: The effect of electrostatic fields on macromolecular synthesis by fibroblasts, in vivo. Abstract. J Cell Biol 39:9, 1968 11. Hunt TK, Van Winkle W: Wound Healing: Normal Repair. South Plainfield, NJ, Chirurgecom, Inc, 1976, pp 1-50 12. Burr HS, Harvey SC, Taffel M: Bio-electric correlates of wound healing. Yale J Biol Med 2:103-107, 1940 13. Shestack R: Handbook of Physical Therapy, ed 3. New York, NY, Springer Publishing Co Inc, 1977, pp 2-23, 40-65 14. Binder SA: Applications of low and high voltage electrotherapeutic currents. In Wolf SL (ed): Clinics in Physical Therapy: Electrotherapy. New York, NY, Churchill Livingstone Inc, 1981, vol 2, pp 1-24
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Fig. 3. Cross sections of skin from treated (A) and untreated (B) rabbits seven days postinjury. The epidermis (E) near the wound site (arrows) is thicker in the treated animal than in the untreated animal. The quantity of new collagen present in the dermis (D) is also greater in the treated rabbit as compared with the untreated rabbit. (H&E stain, 47 x before reduction.)
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