Effect of electrically induced muscle contractions on posttraumatic edema formation in frog hind limbs.

Edema formation is a natural physiologic response to trauma. If allowed to occur unchecked, however, it may produce subsequent functional deficits. Physical therapists have attempted to inhibit posttraumatic edema by using treatments, or a combination of treatments, that include ice, compression, elevation, skeletal muscle contractions, and electrical stimulation.

On recently has the effect of electrical stimulation on edema been demonstrated. [1-4] Though a multitude of electrical stimulators are available, most having numerous options of stimulation characteristics, high voltage pulsed current (HVPC) has received the most attention in regard to its effect on edema. A series of studies recently completed in this laboratory evaluated the effects of 120-pulse per second (pps) cathodal HVPC, at intensities 10% below motor thresholds, on edema formation in frog hind limbs. [1-4] In these studies, HVPC was applied to one hind limb after both hind limbs were uniformly traumatized. Limb volumes were measured via water displacement over a 24-hour period. In each investigation, HVPC was found to limit edema formation in treated limbs relative to untreated control limbs. These studies were the first to provide evidence that "sensory-level" HVPC has an inhibitory effect on edema formation.

An increase in venular wall permeability to plasma proteins is a primary mechanism of edema formation following trauma. [5-7] Reed [8] suggested that HVPC is capable of reducing microvessel permeability to macromolecular proteins and thereby may retard edema formation. High voltage pulsed current, however, may also be used to induce muscle contractions. The "muscle pump" or "venous pump" activity dramatically reduces venous pressure in the feet of walking humans. [9] Muscle contractions cause compression of valved venous and lymphatic vessels. This compression forces fluids proximally, thereby encouraging venous and lymphatic return. [9] High voltage pulsed current, therefore, at intensities above motor threshold, may curb edema formation by inducing repeated muscle contractions to pump fluids out of an injured limb. The purpose of this study was to test the hypothesis that HVPC-induced muscle contractions would curb posttraumatic edema formation in frog hind limbs.

Rationale for Frog Model

Impact injuries and other edema-causing injuries are common in humans. Such 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 to 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 the 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-pumping 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.

Although HVPC is commonly applied to control edema, we have chosen not to model our study protocols after any published clinical method, in part, because no clinical protocol has been demonstrated to be effective. Not only are present clinical protocols unsubstantiated, but such variables as duration of clinical treatments for controlling edema with HVPC seem arbitrary and based more on exigencies of patient scheduling than demonstrated optimal treatment effects. Most clinical protocols using HVPC also include the application of ice, elevation, and compression. Inclusion of any of these standard techniques in our protocols would make it impossible to determine the efficacy of HVPC alone.

In all of the studies performed in our laboratory, we placed frogs in slings to simplify handling and to provide a means of uniformly supporting the frogs during plethysmography, treatment, and rest in a way that rendered all four limbs pendant (ie, in a dependent position). Hind limbs were required to be in a dependent position at least for plethysmography and treatment, because the hind limbs were immersed in order to determine volume displacement and to administer treatment. Such positioning alone is known to increase limb volumes in frogs. [10] Therefore, it seems reasonable to expect dependent positioning to exacerbate posttraumatic edema formation.

In our model, both feet of each frog are traumatized equally so that net increases in limb volumes include swelling induced by trauma as well as positioning. Whereas dependent positioning is not used in conventional clinical practice, orienting the limbs of our frogs in virtually any other position might be construed as therapeutic. Efficacy of HVPC for edema control might be underestimated using our current model, because dependent positioning may mask a small, but positive, treatment effect.

As stated previously, we applied our electrical stimulation via the use of the "immersion technique." This method is used clinically, but we selected it because it eliminates problems associated with surface electrodes (eg, uneven contact with skin, particularly on the small, irregularly shaped body parts of small vertebrates). This method also eliminates difficulties in applying electrodes for long periods, especially under the conditions of our studies. We kept frogs moist and anesthetized by dripping an anesthetic solution over them during rest periods between treatments. Moreover, measuring limb volumes by plethysmography requires repeated immersion in water. Such repeated exposure to moisture would likely cause dissolution of electrode gel/adhesive, and reapplication of surface electrodes would require frequent manipulation of injured limbs and potentially affect limb volumes.

Materials and Methods

Subjects

Twelve bullfrogs (Rana catesbeiana), weighing between 307 and 540 g ([unkeyable]=458, SD=26), 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.

Instrumentation

Instrumentation in this study was identical to that described previously by Taylor et al. [4] 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- X 2-cm sheet of Plexiglas [R] (*) was positioned between the weight and the limb to avoid skin disruption upon 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 libms 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 and weighed on an analytical balance. (1) One gram of water represented 1 mL of limb volume.

The HVPC was supplied by an Intelect 500S stimulator (2) that delivered twin monphasic pulses of 5 and 8 microseconds' duration separated by an interpulse delay of 75 microseconds. Continuous cathodal stimulation at a frequency of one of these pulse pairs per second was applied via the immersion technique using room-termperature tap water. Self-adhering carbon-rubber electrodes (3) (25 X 76 mm) functioned as anodes and were applied to each hind limb over the posterior surfaces of the frogs' proximal thighs, and 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 the malleoli were submerged during treatment).

Procedure

Each fro 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 he anesthetic to 1 L of water. Anesthesia was maintained by soaking the cloth slings in the anesthetic, which was absorbed through the frog's skin. Data were expressed as changes in limb volume per kilogram of body weight. Pre-trauma limb volumes were measured via plethysmography, and hind limbs were traumatized by impact. Immediate postrauma limb volumes were then measured.

Next, both hind limbs were immersed to their premarked points in separate beakers. Four 30-minute treatments of continuous 1-pulse per second (pps) HVPC were applied to one randomly selected limb, with 1-hour rest periods between treatments. Stimulation intensity was increased in the treated limb until a muscle contraction resulting in minimal joint movement was induced. Combined flexion of the hip, knee, and ankle was typically observed. Voltage was adjusted as necessary to maintain minimal joint movement throughout each 30-minute treatment session. The contralateral limb was immersed, but did not receive treatment; thus, it served as a control. The investigator who measured limb volumes (KT) was uninformed regarding limb assignment (treatment or control). Both limbs were in a dependent position during the four treatments and intervening rest periods. Animals were then positioned prone for the remainder of data collection, except for brief periods when the limbs were measured.

In addition to the immediate pretrauma and posttrauma measurements, limb volumes were measured before and after each treatment and at 8, 17, 20, and 24 hours following trauma. Prior to sacrifice, the traumatized area was examined through a skin incision to ensure volume changes resulted from edema formation and not frank bleeding.

Data Analysis

In order to control for minor asymmetry in the frogs' hind limbs, data were expressed as changes in limb volume from petrauma volumes and normalized per kilogram of body weight (mL/kg). Th hypothesis that treated limb volumes would increase less than untreated limb volumes was tested by an ANOVA for repeated measures.

Results

Repeated muscle contractions, as induced in this study, did not limit posttraumatic edema formation in the frogs' hind limbs (Figure). Volumes of both treated and untreated limbs increased significantly (P = .0001) immediately following impact injury (Tabs. 1 and 2, Figure). The difference between treated limb volumes and untreated limb volumes over the 24-hour period following trauma, however, was not significant (P = .85) (Tabs. 1 and 2, Figure). This obvious absence of treatment effect prompted us to cease data collection after 12 animals.

[TABULAR DATA OMITTED]

Discussion

Because four 30-minute treatments of HVPC, which induced repeated twitch contractions at 1 pps, resulted in no significant differences between volumes of treated and untreated limbs, we rejected our hypothesis. We do not interpret this to mean that the muscle-pump technique, a time-honored method of inhibiting edema, is ineffective, but that HVPC as applied in this study resulted in no significant net effect on limb volumes during a 24-hour period following trauma.

Contractions of lower-extremity muscles in standing humans can reduce venous pressure in the feet from

[TABULAR DATA OMITTED]

90 to 20 mm Hg.[9] This decrease in venous pressure can be attributed to decreased venous blood volume achieved by muscular contractions that force fluids proximally in these valved vessels. Though repeated muscle contractions are used to encourage venous and lymphatic flow rates, [11,12] it is possible that the twitch contractions induced in this study were not of sufficient force or duration to create a significant muscle-pump effect. Our decision to use 1-pps stimulation was based on pilot work that indicated that continuous stimulation at higher pulse rates produced profound fatigue (unresponsiveness to stimulation) prior to completion of the fourth treatment session. It is possible that brief, higher-frequency pulse trains may have resulted in a more effective muscle-pump effect.

Though we determined previously that one 30-minute treatment of sensory-level HVPC inhibited edema formation, [4] we applied four 30-minute treatments in this study with the intention of ensuring that treatment would be aggressive enough to elicit any potential treatment effects. No demonstrable treatment effect was present after the first treatment or after any of the subsequent treatments.

A single 30-minute treatment of cathodal HVPC, at 120 pps and intensities 10% less than motor thresholds, has been shown to significantly limit posttraumatic edema formation for more than 4.5 hours, [4] and a series of four 30-minute treatments limited edema formation for up to 24 hours. [2] Such stimulation may inhibit edema by reducing microvessel permeability to plasma proteins. [8] Though HVPC was applied at sufficient intensities in this study (ie, above motor thresholds), a pulse frequency of only 1 pps was used. Because such low frequency current was applied, it is unlikely that significant alterations in microvessel permeability were achieved. Hence, it is unlikely that mechanisms related to microvessel permeability significantly contributed to our results.

Muscle contractions, whether voluntary or electrically induced, will cause an increase in arteriolar blood flow because of increased metabolic demands. [13,14] When muscle contractions cease, the mechanical effects of muscle-pump activity cease abruptly, but increased arteriolar flow persists for several minutes. Increased arteriolar flow continues to allow replenishment of nutrients and energy stores, as well as removal of metabolic byproducts. This increased flow, in pendant human limbs, can return venous pressure to 90 mm Hg within 30 seconds following the completion of exercise. [9] The combination of increased arteriolar flow and an increased venous pressure will result in greater efflux of fluids at the arteriolar end of a capillar bed and an inhibition of reabsorption at the venular end. Such an imbalance may result in a net increase in interstitial fluids (ie, edema). [9,13] Thus, it may have been the case that, though a beneficial musclepump effect existed during the induced exercise, its influence was negated by an increase in arteriolar blood flow and the gravitational effects associated with 5 hours of pendant positioning.

Immediately following trauma, histamine is released, causing an increase in microvessel permeability. Indeed, this increase in permeability is regarded as an essential factor in acute edema formation. [5-7] In the presence of increased microvessel permeability, edema may form even when normal arteriolar pressures exist. Therefore, increased arteriolar flow, as might attend muscle contractions, may result in even greater efflux of plasma proteins through venular walls, particularly in the acute posttraumatic phase. This outward flow of proteins into the interstitial space would tend to exacerbate swelling. We may have seen a demonstrable treatment effect had HVPC treatment been applied later in the inflammatory process, when the balance of forces across the microvascular walls had reached a more stable state.

The problem of increased arteriolar flow, coupled with increased vessel wall permeability, may have been compounded by pendant positioning of the hind limbs. In this study, limbs were in a pendant position a total of 5 hours immediately following trauma (ie, during four 30-minute treatments and three intervening 1-hour rest periods). Though this period of pendant positioning was necessary to allow limb-volume measurement and avoid difficulties of surface electrode attachment, such positioning would tend to promote the accumulation of fluids in distal aspects of limbs. It may be simply that pendant positioning had a greater influence in promoting edema formation than HVPC-induced msucle contractions had in inhibiting edema.

Examination of alternate pulse rates, pulse durations, and intensities is necessary to determine whether HVPC-induced muscle contractions can control edema formation or reduce extant edema. Furthermore, investigation into the relative influences of limb position and HVPC is necessary.

Conclusion

Four 30-minute treatments of 1-pps HVPC that induced repeated msucle contractions (ie, muscle-pump activity) did not significantly limit edema formation in frog hind limbs.

References

[1] 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.

[2] Bettany JA, Fish DR, Mendel FC. The effect of high voltage pulsed direct current on edema formation following hyperflexion injury. Arch Phys Med Rebabil. 1990;71:677-681.

[3] Bettany JA, Fish DR, Mendel FC. Influence of cathodal high voltage pulsed current on acute edema. J Clin Electrophysiol. In press.

[4] Taylor K, Fish DR, Mendel FC, Burton HW. Effect of a single 30-minute treatment of high voltage pulsed direct current on edema formation in frog hind limbs. Phys Ther. In press.

[5] Grega GJ, Adamski SW, Dobbins DE. Physiological and pharmacological evidence of the regulation of permeability. Federation Proceedings. 1986;45:96-100.

[6] Miller FN, Sims DE. Contractile elements in the regulation of macromolecular permeability. Federation Proceedings. 1986;45:84-87.

[7] Cotran RS, Majno G. A light and electron microscopic analysis of vascular injury. Ann NY Acad Sci. 1964;116:750-764.

[8] Reed BV. Effect of high voltage pulsed electrical stimulation on microvascular permeability to plasma protiens: a possible mechanism in minimizing edema. Phys Ther. 1988;68:491-495.

[9] Guyton AC. Textbook of Medical Physiology. 5th ed. Philadelphia, Pa: WB Saunders Co; 1976.

[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] 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.

[12] Ladd MP, Kottke FJ, Blanchard RS. Studies of the effect of massage on the flow of lymph from the foreleg of the dog. Arch Phys MEd. 1952;33:604-612.

[13] Richardson D, Shewchuk R. Effects of contraction force and fequency on postexercise hyperemia in human calf muscles. J Appl Physol: Respirat Environ Exercise Physiol. 1980;49:649-654.

[14] Currier DP, Petrilli CR, Threlkeld AJ. Effect of graded electrical stimulation on blood flow to healthy muscle. Phys Ther. 1986;66:937-943.

(*) Rohm & Hass Co, Independence Mall W, Philadelphia, PA 19105.

(1) Mettler Instrument Corp, 29 Nassau St, Princeton, NJ 08520.

(2) Chattanooga Corp, PO Box 4287, Chattanooga, TN 37405.

(3) Tenzcare, 3M Corp, Bldg 225-5S-01, 3M Center, St Paul, MN 55144-1000.