Effect of a single 30-minute treatment of high voltage current on edema formation in frog hind limbs.

Effect of a Single 30-Minute Treatment of High Voltage Pulsed Current on Edema Formation in Frog Hind Limbs

The purpose of this study was to investigate the effect of a single treatment of high voltage pulsed current (HVPC) on edema formation. Twenty-four frogs were anesthetized, and both hind limbs of each frog were traumatized by impact. Limb volumes were measured by water displacement immediately before and after trauma and at predetermined intervals for 24.0 hours posttrauma. One limb of each frog was randomly selected to receive 30 minutes of continuous, 120-pulse per second, cathodal HVPC at voltages 10% less than motor threshold levels. Data were analyzed by an analysis of variance for repeated measures. Sources of significant differences were determined by paired t test (probability values determined by Bonferroni adjustment). A single 30-minute application of HVPC significantly curbed edema formation for between 4.0 and 7.5 hours following treatment (ie, volumes of treated limbs were significantly less than those of untreated limbs). These results suggest that regimens currently applied to humans (ie, one treatment per day or three times per week) may be insufficiently aggressive to provide sustained treatment effects. [Taylor K, Fish DR, Mendel FC, Burton HW. Effect of a single 30-minute treatment of high voltage pulsed current on edema formation in frog hind limbs. Phys Ther. 1992;72:63-68.]

Key Words: Edema, Electrical stimulation, High voltage pulsed current, inflammation. Following traumatic injury, an acute local inflammatory response occurs that follows a relatively predictable sequence of events.[1] One such event is the formation of edema. Though edema is a natural consequence of tissue injury, it often leads to pain and loss of function of the injured body part. Curbing the excessive accumulation of interstitial fluid to limit negative sequelae is therefore a typical aim of physical therapy. Although a time-honored treatment regimen of ice, compression, and elevation has been used to control edema, performing such a regimen may interfere with necessary activities of daily living. It is now common practice for therapists to apply an alternative treatment, high voltage pulsed current (HVPC), to control edema.[2-4] The effectiveness of HVPC in controlling edema, however, has not been fully established. Mohr et al[5] reported no significant treatment effect of HVPC on posttraumatic edema in rat hind limbs. Reed,[6] however, stated that HVPC may reduce microvascular permeability and thus may be beneficial in controlling edema. Bettany et al[7] also examined the effect of HVPC on edema formation. Both hind limbs of 20 bullfrogs (Rana catesbeiana) were traumatized by impact. One hind limb received four 30-minute treatments of 120-pulse per second (pps) cathodal HVPC at voltages 10% below motor thresholds; contralateral limbs received no stimulation. This aggressive HVPC treatment regimen effectively limited edema formation.[7] Further studies[8,9] confirmed that HVPC reduces edema formation after simulated ankle sprain and when treatment was delayed for 4.5 hours following impact injury. Although the literature provides good reason to hypothesize the utility of aggressive application of HVPC (ie, four 30-minute treatments or one 6-hour treatment) in limiting edema formation,(7-9) the effect of a common clinical application, a single HVPC treatment applied after injury, is unknown. Thus, the purpose of this study was to determine the duration of any significant treatment effect resulting from a single 30-minute treatment of 120-pps cathodal HVPC and to corroborate or refute the findings of Bettany and colleagues.[7-9] We hypothesized that treated limb volumes would increase less than untreated limb volumes.

Rationale for Frog Model

Impact injuries and other edema-causing injuries are common in humans. Sucn naturally occurring injuries vary in severity and anatomical location, however, and they occur in people who differ in age, sex, health, and a host of other variables. This variability mandates that studies of the effects of HVPC on edema in humans have a large sample size to reduce the confounding influence of variability in such factors as severity of injury, site of injury, age, sex, health, and time elapsed from injury to initiation of treatment. Such studies are expensive and time-consuming. To better control variables and reduce time and costs, nonhuman models, namely rats, have been used extensively to investigate the effectiveness of various methods of treating edema. Small mammals, including rats, do not readily tolerate prolonged anesthesia. The high metabolic rates of small mammals also preclude prolonged anesthesia without intravenous feeding. Furthermore, heat and water loss are difficult to control without tampering directly with vascular systems of small mammals. Such interference may well affect activities at the capillary level. If anesthetized just for trauma, the animals must either be restrained after they awaken, a stressful situation for most animals, or allowed free movement, which may itself affect limb volumes by way of displacing fluids via "muscle-pump" activity. Free movement may also subject the animal to pain and the injured body part to further injury or manipulation (eg, massage, licking), which may affect limb volumes. Limb volumes must be determined on relaxed, perfectly still limbs (ie, on anesthetized animals), and treatment, especially sensory-level electrical stimulation, is delivered more effectively and with less evoked stress when the animal is anesthetized. Repeated use of anesthetics within 1 day or even once a day, however, often results in death. To avoid some problems associated with human subjects or the use of small mammals, we chose bullfrogs as the experimental model for this study on edema. Bullfrogs can be anesthetized through the skin for an entire day without interdicting their vascular system. Pain, stress, and movement (muscle-pumping activity) are eliminated as confounding factors. Repeated limb-volume measurements are easily accomplished, and strong sensory-level treatments are administered without inducing stress or discomfort. Water loss is easier to control in amphibians than in small mammals. Because amphibians absorb water through their skin, simply keeping them moist effectively negates water loss. Anesthesia, of amphibians or mammals, slows heart rate and probably most other physiologic responses, including responses to trauma. Observations of traumatized anesthetized animals (amphibians or mammals), however, indicate that fairly typical physiologic responses do occur,[10] albeit at a rate that may be different from that of fully alert animals. Such observations also indicate that the physiologic responses for each class of vertebrate are similar in kind and in function and therefore that their physiologic processes are probably similar.

Method

Subjects

Twenty-four bullfrogs (Rana catesbeiana), weighing between 352 and 483 g (X [Bar] = 421, SD = 18.6), were used in this study. All procedures relating to the care and handling of the animals were approved by the Institutional Laboratory Animal Care Committee of the State University of New York at Buffalo prior to initiating data collection.

Instrumentation

Instrumentation followed that described by Bettany and colleagues,[7-9] with minor modifications. Both feet of each frog were traumatized by dropping a 352.5-g steel bar from a height of 66 cm onto the plantar aspect of each foot. This steel bar fell through a cylindrical tube and struck each foot just distal to the malleoli. A 2-X2-cm sheet of Plexiglas [R]* was positioned between the weight and the limb to avoid skin disruption on impact. Because frog body weight varied, dropping the same weight from a uniform height did not inflict uniform injuries across the group; smaller animals received greater injuries than larger animals. We chose not to normalize impact forces relative to body weight, because each animal served as its own control. Our design requires only the assumption that treated limbs as a group are injured to the same extent as untreated limbs. Both feet of all animals were traumatized as uniformly as possible, and the treated limbs were compared statistically with the contralateral untreated limbs, which were handled in precisely the same way as treated limbs except for stimulation. Limb volumes were determined via plethysmography. The plethysmograph consisted of a large glass immersion vessel that was filled with distilled water and connected to an overflow chamber by rubber surgical tubing. Glassware was thoroughly cleaned with soap and water twice during each treatment session to minimize adherence of water droplets to the overflow chamber, thus maximizing measurement reliability. Reliability of this limb-volume measurement system was tested by performing 30 repeated limb-volume measurements on each of four untraumatized frog hind limbs. Estimation of reliability using analysis-of-variance (ANOVA) procedures yielded an intraclass correlation coefficient of .995. Actual variation between maximum and minimum limb-volume measurements for the four limbs was only 0.6 to 1.1 mL. During reliability testing and throughout the actual data-collection process, each frog was suspended in a cloth sling. We could thereby move frogs without handling their limbs. Each frog was lowered by an adjustable camera boom until the limb to be measured was submerged into the immersion vessel to a premarked point on the frog's calf. Water displaced by limb immersion was collected in a 100-mL beaker. The weight of displaced water was measured on a digital scale.([dagger]) One gram of water represented 1 mL of limb volume. The HVPC was supplied by an Intelect 500S stimulator([double dagger]) that delivered twin monophasic pulses of 5 and 8 microseconds' duration separated by an interpulse delay of 75 microseconds. Continuous cathodal stimulation at 120 pps was applied via the immersion technique using room-temperature tap water. Self-adhering carbon-rubber electrodes([S]) (25 X 76 mm) functioned as anodes and were applied to each hind limb over the posterior surfaces of the frogs' proximal thighs; these anodes remained out of the water. Cathodes, also self-adhering electrodes, were applied below the water's surface to the inside walls of the treatment beakers at a depth of 8 cm (ie, the level to which malleoli were submerged during treatment).

Procedure

Each frog was anesthetized prior to trauma by 20 to 30 minutes of immersion in an aqueous solution of MS222 (3-aminobenzoic acid ethyl ester), prepared by adding 3 g of the anesthetic to 1 L of water. Anesthesia was maintained by soaking the cloth slings in the anesthetic, which was absorbed through the frogs' skin. Data were expressed as changes in limb volume per kilogram of body weight. Dots were painted on the frogs' legs 2 cm distal to the fibular heads, and pretrauma limb volumes were measured via plethysmography. Hind limbs were then traumatized by impact, and limb volumes were determined immediately posttrauma. Both hind limbs were immersed to their premarked points in separate beakers. A 30-minute HVPC treatment was applied to one (randomly selected) limb; the other limb was immersed, but did not receive treatment. Stimulation intensity was increased in the treated limb until a minimal muscle contraction was observed. Then, using an oscilloscope([//]) as a monitor, voltage was reduced by 10%. Voltages between 30 and 40 V were applied, yielding pulse charges of 0.75 to 1.0 [Mu] C, respectively. Treatment intensity was maintained at this voltage throughout the 30-minute treatment session. The control limb received sufficient stimulation to produce one minimal muscle contraction, after which stimulation was immediately terminated. This contraction was induced in control limbs to match the brief contraction induced in treated limbs, thereby eliminating the possibility of differential muscle-pump effects. We chose this stimulation intensity (ie, 90% of that motor threshold) intending to be as aggressive as possible without invoking a muscle-pump effect. Animals were positioned prone for the remainder of the 24-hour posttrauma period, with the exception of brief periods when limb volumes were measured. In addition to the immediate pretrauma and posttrauma measurements, limb volumes were measured immediately following treatment and at 1.5, 3.0, 4.5, 8.0, 17.0, 20.0, and 24.0 hours following trauma. Data were collected by an investigator (KT) who did not know the treated and control limb assignments. Prior to sacrifice, the traumatized area was examined through a skin incision. Hematomas were found in limbs of three animals; associated data were eliminated from the data analysis because the purpose of the study was to evaluate the effect of HVPC on edema formation. Gross disruption of vascular beds and frank bleeding may have influenced mechanisms of action of HVPC in these frogs.

Data Analysis

Data were expressed as changes in limb volumes from pretrauma volumes and normalized per kilogram of body weight. The hypothesis that treated limb volumes would increase less than untreated limb volumes was tested by two-way ANOVA for repeated measures on two factors (time and treatment group). Sources of significant differences were determined by paired t tests specific to each repeated measure. Because nine such comparisons were performed, we required a more stringent level of significance (P < .0056). This level of significance was determined by applying the Bonferroni correction, which involved dividing .05 (the initial level of significance) by 9 (the number of repeated measures).[11]

Results

The single 30-minute HVPC treatment significantly curbed edema formation for at least 4 hours following treatment. The ANOVA revealed that, although volumes of both treated and untreated limbs increased in the posttraumatic period (Figure), the volume increase in treated limbs was significantly lower (P=.003) than the increase in untreated limbs (Tab. 1, Figure). Repeated tests indicated that changes in volumes of treated limbs were significantly less than those of untreated limbs from immediately after treatment through 4.5 hours posttrauma (ie, 4 hours after treatment ended) (Tab. 2, Figure). Thereafter, volumes of treated limbs were not significantly different from those of untreated limbs (Tab. 2, Figure).

Discussion

One 30-minute treatment of 120-pps cathodal HVPC delivered at a voltage 10% less than visible motor threshold significantly inhibited edema formation in the frogs' hind limbs. These results corroborate the reports of Betany and colleagues[7,8] that HVPC curbs posttraumatic edema. In those studies, four 30-minute HVPC treatments were applied to one (randomly selected) frog hind limb and the other limb served as a control. This regimen of four treatments curbed edema formation from the end of the first treatment to 8 hours following impact injury and through 24 hours following hyperflexion injury. In both of these studies, a significant difference in edema formation was noted between treated and untreated limbs after the first treatment session, but the authors were unable to determine the duration of the effect of the first treatment, because three subsequent treatments were applied during data collection. We have established that a single 30-minute HVPC treatment curbs edema formation and that the treatment effect is finite, lasting between 4.0 and 7.5 hours following treatment. Generalization of these data, gathered from amphibians, to humans must be done with extreme caution. Our model, like any other, has limitations. Frogs and humans, as fellow vertebrates, share significant and fundamental physiologic characteristics. In physiologic responses germane to our study, amphibians have long been used as models for humans. Indeed, many basic tenets of electrotherapy have been determined on amphibians, frogs in particular, and applied to mammals, humans in particular, with good fidelity. We believe, therefore, that the results of our study of frogs are suggestive of those to be expected in studies of human subjects. If we assume that these results at least may serve as a basis for clinical hypothesis, it would seem insufficient to apply HVPC in the common schedule[2-4] of one treatment per day or three times per week. This and previous studies[7-9] performed in our laboratory suggest that a much more aggressive regimen, perhaps one 30-minute treatment every 4 hours, may be required to sustain clinical treatment effects. Multiple daily HVPC treatments could be performed by patients using portable electrical stimulators. Stimulation might simply be turned on and off throughout the day without disrupting daily activities. The mechanism of action of HVPC and the relative effectiveness of HVPC versus other traditional treatments (ie, ice, compression, and elevation) have yet to be thoroughly investigated. Increased permeability of postcapillary venules to plasma proteins is the predominant mechanism by which post-traumatic edema occurs.(12-14) As excessive plasma proteins move into the interstitial space, fluid may be drawn out of the vascular bed because of tis imbalance of protein across the vessel wall, resulting in edema formation. Reed(6) theorized that HVPC may be effective in reducing edema formation by inhibiting the leakage of plasma proteins from the vascular bed. The influence of current polarity is also unknown. Cathodal stimulation as applied in our study may be more effective in altering membrane permeability than a similar treatment of anodal stimulation. It seems unlikely, however, that charge repulsion (ie, negative charge repelling negatively charged proteins from the edematous area) was significant in this study, as pulse duration was very short and pulse charge was quite low. It is reasonable to surmise that cryotherapy also limits swelling via alterations in the balance of forces across plasma membranes. Cold application reduces membrane permeability and arteriolar blood flow and thus may limit edema formation.[15] Therapists must use caution, however, when applying or instructing their patients to apply cryotherapy. Excessive cooling may result in tissue ischemia and therefore tissue damage. No such precautions are involved with the application of HVPC. Furthermore, as with transcutaneous electrical nerve stimulation for pain control, HVPC treatment variables may be preset on home units to promote safe stimulation in the therapist's absence. The second component of the traditional treatment approach, compression, influences edema formation by increasing interstitial pressure. Such a pressure increase may reduce efflux of fluid and plasma proteins from the microcirculation, thereby inhibiting edema formation.[1] Caution must be used when applying compressive wraps, ensuring that external pressure does not compromise blood flow to the point of issue ischemia. It is sometimes difficult, however, to determine the pressure at which deleterious effects may occur. Elevation, the third component, is simply positioning an injured limb such that gravity encourages venous and lymphatic flow out of traumatized area.(15) Elevation of extremities is often impossible, however, during daily activities. In contrast, HVPC tretment could be applied safely and easily via portable units worn during such daily activities. Each of these treatments (ice, compression, elevation, and HVPC) may be beneficial in inhibiting edema formation. Further investigation of the relative effectiveness of each individual treatment as well as its potential additive effects, however is necessary. Continued efforts are being made in our laboratory to elucidate mechanisms of action and specific effects of frequency, polarity, waveform, and intensity on physiologic responses to electrotherapy.

Conclusion

A single-30-minute treatment of cathodal HVPC at 120 pps and a voltage 10% less than that necessary to produce a minimal visible muscle contraction inhibited acute edema formation in frog hind limbs following impact injury in this study. Volumes of treated limbs remained significantly less than those of untreated limbs for between 4 and 7.5 hours after treatment. These results suggest that regimens currently applied to humans (ie, one treatment per day or three times per week) may be insufficiently aggressive to provide sustained treatment effects.

Table 1. Analysis-of-Variance Results for Repeated Measures on Two Factors

Source                  df   SS        MS       F       P(a)
Limb assignment (A)     1     461.35   461.35   11.32   .003
Repeated measures (B)   8    5323.71   665.46   37.86   .0001
A x B                   8     108.99    13.62    3.04   .003

(a)P<.05.

Table 2. Results of Post Hoc Paired t Tests for Posttrauma Comparisons of Treated and Untreated Limb-Volume Measurements

Measurement               df   Mean X-Y(a)   Paired t   P
Immediate posttrauma      20   1.3           1.431      .084
Immediate posttreatment   20   3.295         3.745      .0007(b)
1.5 h posttrauma          20   3.5           3.012      .0034(b)
3.0 h posttrauma          20   3.333         3.689      .0007(b)
4.5 h posttrauma          20   3.024         3.72       .0007(b)
8.0 h posttrauma          20   1.72          2.379      .0137

(a)X = change in untreated limb volume (in milliliter per kilogram of body weight); Y = change in treated limb volume (in milliliters per kilogram of body weight). (b)Significant at P < .0056, calculated using Bonferroni adjustment (P = .05/9 repeated t tests).

(*)Rohm & Haas Co, Independence Mall W, Philadelphia, PA 19105. ([dagger])Mettler Instrument Corp, 29 Nassau St, Princeton, NJ 08520. ([double dagger])Chattanooga Corp, PO Box 4287, Chattanooga, TN 37405. ([S])Tenzcare, 3M Corp, Bldg 225-5S-01, 3M Center, St Paul, MN 55144-1000. ([//])Tektronix, PO Box 500, Beaverton, OR 97077.

PHOTO : Figure. Mean limb-volume changes and standard errors of treated and untreated limbs over time. (Asterisk indicates significant difference between groups.)

References

[1]Guyton AC. Textbook of Medical Physiology 5th ed. Philadelphia, Pa: WB Saunders Co; 1976. [2]Ralston DJ. High voltage galvanic stimulation: Can there be a state of the art? Athletic Training. Winter 1985: 291-293. [3]Voight ML. Reduction of posttraumatic ankle edema with high voltage pulsed galvanic stimulation. Athletic Training. Winter 1984:278-311. [4]Sorenson MK. Galvanic Stimulation for Post-op Edema in the Hand. American Physical Therapy Association Section on Clinical Electrophysiology; April 1984. [5]Mohr TM, Akers TK, Landry RG. Effect of high voltage stimulation on edema reduction in the rat hind limb. Phys Ther. 1987;67:1703-1707. [6]Reed BV. Effect of high voltage pulsed electrical stimulation on microvascular permeability to plasma proteins: a possible mechanism in minimizing edema. Phys Ther. 1988;68:491-495. [7]Bettany JA, Fish Dr, Mendel FC. Influence of high voltage pulsed direct current on edema formation following impact injury. Phys Ther. 1990;70:219-224. [8]Bettany JA, Fish DR, Mendel FC. The effect of high voltage pulsed direct current on edema formation following hyperflexion injury. Arch Phys Med Rehabil. 1990;71;677-681. [9]Bettany JA, Fish DR, Mendel FC. Influence of cathodal high voltage pulsed current on acute edema. J Clin Electrophysiol. In press. [10]Fish DR, Mendel FC, Bettany JA. Research design considerations in studying edema. Phys Ther. 1990;70:76-78. Author response to letter to the editor. [11]Nemenyi P, Dixon SK, White NB, Hedstrom ML. Statistics from Scratch. San Francisco, Calif: Holden-Day; 1977. [12]Grega GJ, Adamski SW, Dobbins DE. Physiological and pharmacological evidence of the regulation of permeability. Federation Proceedings. 1986;45:96-100. [13]Miller FN, Sims DE. Contractile elements in the regulation of macromolecular permeability. Federation Proceedings. 1986;45:84-87. [14]Cotran RS, Majno G. A light and electron microscopic analysis of vascular injury. Ann NY Acad Sci. 1964;116:750-764. [15]Knight KL. Cryotherapy: Theory, Technique, and Physiology. Chattanooga, Tenn: Chattanooga Corp; 1985.

K Taylor, MS, PT, is Assistant Professor, Department of Physical Therapy, Daemen College, 4380 Main St, PO Box 784, Amherst, NY 14226 (USA). This study was completed in partial fulfillment of the requirements for Mr Taylor's Master of Science degree in exercise science at the State University of New York at Buffalo. Address all correspondence to Mr Taylor. DR Fish, PhD, PT, is Associate Professor, Department of Physical Therapy and Exercise Science, and Director, Electrotherapy Research Laboratory, State University of New York at Buffalo, 411 Kimball Tower, Buffalo, NY 14214. FC Mendel, PhD, is Associate Professor, Department of Anatomical Sciences, State University of New York at Buffalo. HW Burton, PhD, is Assistant Professor, Department of Physical Therapy and Exercise Science, State University of New York at Buffalo. The results of this research were presented at the Annual Conference of the American Physical Therapy Association; June 23-27, 1991; Boston, Mass. This study was approved by the Institutional Laboratory Animal Care Committee of the State University of New York at Buffalo. This study was supported by a Diamond Research Fund Grant from the Graduate Student Association, State University of New York at Buffalo.