The electrical effect of two commonly used clinical stimulators on traumatic edema in rats.

Key Words: Animal; Edema; Electrotherapy, electrical stimulation; Rats,. Wounds and injuries.

In recent years, the application of pulsatile electrical stimulation for the resolution of traumatic edema has become widespread. Unfortunately, no controlled clinical study has provided evidence to demonstrate the efficacy of electrical stimulation in reduction of edema.(1) In a number of clinical reports,(2-6) investigators have reportedly observed that electrical stimulation reduced traumatic edema. In contrast, Mohr et al(7) concluded that following trauma, monophasic pulsed electrical stimulation did not cause reduction of rat paw edema in treated animals. Bettany and colleagues(8-10) have recently demonstrated that swelling of the sprained frog ankle can be retarded by high voltage stimulation using a monophasic waveform, but the effect occurred only during, not between, stimulation sessions. They suggested that longer application of electrical stimulation may result in more significant edema reduction.

The absorption mechanism, which electrical stimulation is presumed to enhance, is hypothetical and based in part on the electrophysical phenomenon described by Alon and DeDomenico.(11) This phenomenon suggests that the passage of electrical current through the tissue electrophysically displaces the negatively charged plasma proteins found in the interstitium of the traumatized areas.(12) This increased mobility should accelerate protein uptake by the lymphatic capillaries, which in turn facilitates lymphatic flow, an established mechanism for extracellular fluid uptake.(13,14) Evidence supporting Alon and DeDomenico's hypothesis was recently presented in an unpublished report by Cook et al (15) when they reported significantly greater lymphatic uptake of proteins in stimulated rats than in a control group. The stimulation paradigm included submotor excitation.(15) Another mechanism that is known to enhance lymphatic flow is the contraction of skeletal muscles and the resultant increase in interstitial pressure.(14) Therefore, in order to study the isolated effect of electrokinetic influences on protein mobility, muscle contraction must be avoided. We believe this can be achieved by limiting the stimulation to submotor excitation.

At the submotor stimulation level, an effective displacement of the proteins into the lymphatic capillaries may depend on the stimulus waveform or the total amount of current passed through the interstitial spaces.(12) Hypothetically, symmetrical biphasic waveforms may not be adequate for fluid reabsorption. Such pulses may not provide a net displacement of proteins in the stimulated area because of the rapid reversal of the current flow direction. Black(16) concurred with the hypothesis that unidirectional current flow is desired for edema reduction, concluding that monophasic waveforms cause direct nonexcitatory effects on charged particles at the cellular level.

Despite the lack of evidence in support of the efficacy of electrical stimulation in edema reduction, clinicians routinely use the modality. They use various waveforms to reduce traumatic edema and, in our opinion, often claim clinical success. Testing these claims seems long overdue.

The purposes of this investigation were (1) to study the effect of electrical stimulation on traumatic rat hind-paw volume and (2) to determine whether two commonly used stimulators differ in their effect on paw volumes. The null hypothesis stated that there would be no difference in paw volumes between (1) the treated and nontreated groups and (2) the two stimulated groups.

Method

Forty-four male Sprague-Dawley rats, initially weighing 200 to 250 g, were used for this study. The rats were randomly assigned to a control group (n=15), a group that received monophasic pulsed current (MPC group, n = 15), or a group that received symmetrical biphasic pulsed current (SBPC group, n=14). The animals were housed in the Animal Facility of the University of Maryland Medical School Teaching Facility under controlled light/dark and temperature conditions, with food and water provided ad libitum.

Before each experimental session, the rats were anesthetized by an intraperitoneal injection of 50 mg of sodium pentobarbital solution per kilogram of body weight. Each rat was secured in a plastic cylindrical restrainer with one open end from which the hind paw was extended. The paw was shaved, and a line was drawn with indelible ink at the level of the medial malleolus.

The restrainer was clamped to a horizontal lever attached to a tripod, which was equipped with a crank that was adjusted to the height of the lever. This arrangement minimized position change when the rat's paw was lowered into the immersion vessel.

All rat paw volumes were measured with a plethysmograph, as described by Mohr and Akers.(17) Modifications of this apparatus involved the substitution of the device's single microsyringe with two Model S-1200A micrometric syringes(*) and the insertion of a bipolar needle(t) into the center of the side arm. When in contact with the solution, the needle, connected to a 60-kHz square-wave 1-V power source,(tt) sent pulses to an oscilloscope (Fig. 1). Therefore, when the proper solution level was reached during a measurement, the oscilloscope would reflect a change in wave amplitude. We believe this method provided a more objective and accurate means of determining the water level than the visual estimates of Mohr and Akers.(17) At the beginning of measurement, the micrometer was set at zero and solution was added to the immersion vessel until the level reached the zero mark on both the vessel and side arm. The extended rat paw was lowered into the immersion vessel until the line on the medial malleolus reached the zero mark (Fig. 2). The displaced solution was then drawn off back to the zero mark, which was confirmed by the change in wave amplitude on the oscilloscope. The displaced volume measured by the micrometer was recorded and designated as the baseline measurement. The paw was removed from the apparatus, and the microsyringe was again set at zero.

The intertester and intratester reliability of the three investigators (SFB, KAC, TLJ) who measured the paw volumes was established by having each tester measure the volume of seven rat paws, with three trials per paw. Testers alternated throughout the trials so that no tester measured paw volumes consecutively. Intraclass correlation coefficients (ICC[2,1])(18) were used to determine the intertester and intratester reliability (ICCs=.99 and .94, respectively).

Following the initial volume measurement, the right hind paw was traumatized. A 50-g weight of 0.5 cm diameter was dropped through a glass tube directly onto the paw from a 50-cm vertical height (Fig. 3). Suckert(19) showed this technique produced the greatest volume increase without causing bone fracture.

Twenty-four hours posttrauma, the paw volume was measured again. Immediately following this measurement, the rats were prepared for stimulation or sham treatment. Self-adhesive surface electrodes,(t) 2.3 x 1. 5 cm in size, were placed on the dorsal and plantar surfaces of the traumatized foot so that the entire swollen area was covered. The electrodes were further secured with tape for uniform electrode contact.

Electrical stimulation was then administered for 1 hour to the rats in the experimental groups using the MPC,(sections of) and SBPC(parallel) stimulators. The settings of the two stimulators are presented in Table 1. The settings for the MPC device were selected to approximate the protocol of Mohr et al.(7) As the phase duration of the stimulator with the monophasic waveform was fixed at 5 to 10 microseconds, the device with the symmetrical biphasic waveform was set at the shortest available phase duration ie, 50 microseconds). During pilot testing, we found that the phase charge at submotor stimulation was about five times lower during SBPC than during MPC. We therefore decided to set the SBPC device's pulse rate at 500 pulses per second so that the total current (in microcoulombs per second) would approximate the total current of the MPC device. We based this strategy on the presumption that protein uptake by the lymphatics may predominantly depend on total current. Amplitude of stimulation was increased until muscle contraction was visible, then decreased slowly to attain submotor stimulation. Peak voltage (MPC) and phase charge (SBPC) were recorded during each stimulation session. The control group was also anesthetized, and a sham treatment was administered by placing electrodes that were not connected to a stimulator over the traumatized paw for 1 hour. At 48 and 72 hours posttrauma, a volume measurement followed by the appropriate 1-hour treatment was repeated so that each rat received a total

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(parallel) Chattanooga corp, 4717 Adams Rd., Hixson, TN 37343 of 3 hours of intervention over a 3-day period. At 96 hours posttrauma, a final volume measurement was obtained.

Results

To ascertain whether significant edema resulted from the traumatization procedure, a 3 x 2 analysis of TABULAR DATA OMITTED variance (ANOVA) for repeated measures on the factor of time was used to compare pretrauma and 24-hour posttrauma paw volumes. The results (Tab. 2) revealed that, at 24 hours posttrauma, all groups had a comparable mean increase in paw volume of approximately 21% (Fig. 4). There were no paw volume differences between the groups at the pretrauma measurement or at the 24-hour posttrauma measurement.

The volumetric measurements at 24 hours posttrauma were considered 100% paw volume, and each of the subsequent measurements at 48, 72, and 96 hours posttrauma were calculated as percentage of change relative to the 24-hour posttrauma data. These percentages of change were subjected to a 3 x 4 ANOVA for repeated measures on the factor of time. The results are summarized in Table 3. Significant differences in paw volume were found between groups, over time, and for the group-time interaction.

A Newman-Keul's post hoc test of the group-time interaction revealed a significantly lower reduction of paw volume in the SBPC group than in the control group at 96 hours posttrauma. There was no significant difference in paw volume, however, between the

Table 2. Two-Factor  Group x
Time) Analysis-of-Variance Results for
Trauma-Induced Paw Volume
Source             df    ms        F
Group               2    0.06      1.64
Error              41    0.04
Time                1    4.42      274.86[sup. a]
Timexgroup          2    0.00         0.80
Error              41    0.02
  [sup.a] Significant at alpha level of .01.

MPC and SBPC groups or between the MPC and control groups at 96 hours posttrauma. Visible edema was still evident in all animals at the last measurement. The post hoc test further revealed a significant reduction in paw volume in the control and MPC groups at 48 hours posttrauma, with no further significant reduction at 72 and 96 hours posttrauma. In contrast, the SBPC group demonstrated significant reduction in paw volume only at 96 hours posttrauma. The means and standard deviations for percentage of reduction in rat paw volume of all groups from 24 to 96 hours posttrauma are illustrated in Figure 5.

On each of the 3 days of stimulation, the mean total current (in microcoulombs per second) administered to the SBPC group was greater than that administered to the MPC group. There was also a consistent reduction of mean total current from day 1 to day 3 in both groups. The means and standard deviations of total current administered to both groups are illustrated in Figure 6. The ICCs for the correlation between volume change and total current were .43 and -.05 for the MPC and SBPC groups, respectively.

Discussion

The results of this study are similar to those reported by Mohr et al,7 who found no significant difference in edema reduction between a control group of rats and a group of rats treated with MPC. Mohr et al suggested that 20 minutes of stimulation may not be adequate to cause any

Table 3. Two-Factor (GroupxTime)
Analysis-of-Variance Results for Percentage
of Reduction of Paw Volume
Source           df     MS      F[sup. a]      Power
Group              2   178.76    3.73          .45
Error             41    47.92
Time               3   486.19   36.85          .99
Timexgroup         6    29.23    2.21          .40
Error            123    13.19
 [sup. a] Significant at alpha level of .05.

significant reduction of traumatic edema. We tripled that duration of stimulation, but still did not demonstrate a significant treatment effect. Further research is needed to determine whether electrical stimulation can significantly enhance edema reduction and, if so, how many hours of stimulation are necessary.

Because the animals in all groups in our study exhibited normal mobility after trauma and were allowed to move freely in their cages throughout the day, muscle activity probably facilitated fluid uptake.(20) Thus, any volume reduction resulting from the MPC treatment may have been masked by the pumping action of the muscles and therefore may not have been detected in our results.

Unlike the unsuccessful attempt by Mohr et al(7) to hasten absorption of extant edema, Bettany and colleagues(8-10) recently reported that the formation of edema in traumatized frog limbs was significantly curbed by high voltage stimulation. The effect was present only during stimulation and for a few hours thereafter. These findings further support the view that 1 hour of stimulation with MPC may not suffice to significantly accelerate edema absorption. Cook et al(15) showed that 1 hour of stimulation with MPC facilitated the uptake of proteins via lymphatics channels, even though they found no reduction of limb volume in an experimental group of rats as compared with a control group of animals. These findings concur with our results and suggest that a 1-hour treatment of MPC was not sufficient to increase the oncotic gradient to a level that will also draw significant fluid from the interstitium.

An additional purpose of this investigation was to test a clinical stimulator (the SBPC device) that, unlike the MPC device, delivers a symmetrical biphasic waveform. The SBPC treatment had a seemingly adverse effect on edema reduction, as evidenced by the significantly lower reduction of edema in the SBPC group versus the control group at 96 hours posttrauma. Unlike the monophasic waveform, the symmetrical biphasic waveform leaves no net charge residue in the interstitium.(11,12) It is possible that the reversing electrical field and the net zero pulse charge created an oscillatory electrokinetic effect. This effect would result in no net movement of the charged proteins, thereby hindering their normal uptake by the lymphatics.

Another factor that potentially interfered with the edema-reduction process was the total current delivered by the SBPC device. Total current is calculated as a product of pulse charge and pulse rate.(12) Thus, many different combinations of pulse characteristics can yield the same total current. As long as the total current is maintained in an appropriate range, the nonexcitatory effect on the proteins will be similar.(21-23) Whether a range of total current critical for edema absorption exists is unknown. Our findings that the SBPC group had a significantly higher total current and that the SBPC group's rate of edema absorption was slower, as compared with the control and MPC groups, imply that relatively high total current or current density may have irritated the edematous tissue and caused smaller decreases in paw volume. Despite efforts to keep the mean total current the same for both stimulated groups, various factors made this variable difficult to control. These factors included stimulator design, varying impedance, and different sensitivity to stimulation among the rats.

The differences in waveform and total current created a confounding effect that precludes a conclusion as to which of the two variables was more accountable for the smaller decrease in paw volume in the SBPC group as compared with the MPC group. Further research is needed to elucidate the questions of preferred waveform and optimal total current for edema reduction.

Other investigations,(7,15) including our own, have used electrical stimulation characteristics similar to those used in the clinic. Although these studies demonstrated no significant enhancement of edema reduction using electrical stimulation, clinicians have reportedly observed favorable results.(2-6) Animal studies and clinical conditions should be compared with much caution. Available animal data on edema reduction have been limited to a few studies and relatively small sample sizes and, therefore, are inconclusive.(7,15,19) Until the data are verified on larger sample sizes, application of these data to human subjects may be premature.

Furthermore, our results may not be directly applied to human subjects because of human subjects' different response to tissue insult. Unlike the rat, which continues to use the injured limb, human mobility and muscle activity are usually limited because of pain or swelling, or both.(24,25) To better simulate a patient response, further animal studies are needed that more effectively restrict the animals' limb movements. By doing so, the isolated effect of electrical stimulation on edema resolution can be better elucidated.

Conclusions

The results of this study demonstrated that the electrical stimulation characteristics and procedures currently used in the clinic do not significantly decrease rat paw volume. Furthermore, the SBPC group demonstrated less decrease in paw volume than did the control group, indicating that a symmetrical biphasic waveform may not be desirable for the purposes of edema reduction. There is a need for future research to possibly find an optimal range of current that would lead to more favorable results. Additional studies should also be conducted to determine whether a longer treatment time is necessary. These future studies would also need to control for any muscle-pumping action that may occur so that any "masking" of the isolated nonexcitatory effect of electrical stimulation is eliminated.

References

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7 Mohr TM, Akers TK, Landry RG, Effect of high voltage stimulation on edema reduction in the rat hind limb. Phys Ther. 1987;67:17031707.

8 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,

9 Bettany JA, Fish DR, Mendel FC. High voltage pulsed direct current: effect on edema formation after hyperflexion injury. Arch Phys Med Rehabil 1990;71:677-681.

10 Bettany JA, Fish DR, Mendel FC. Influence of cathodal high voltage pulsed current on acute edema. J Clin Electrophysiol 1990;2:5-8.

11 Alon G, DeDomenico G. High Voltage Stimulation. Chattanooga, Tenn: Chattanooga Corp; 1987:129-146.

12 Alon G. Principles of electrical stimulation. In: Nelson RM, Currier DP, eds. Clinical Electrotherapy. 2nd ed. East Norwalk, Conn: Appleton & Lange; 1991:35-103.

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15 Cook H, Morales M, LaRosa E, et al. Effects of electrical stimulation on lymphatic flow and limb volume in the rat. Presented at the Annual Conference of the American Physical Therapy Association; June 24-28, 1990; Anaheim, Calif.

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17 Mohr TM, Akers TK. Simplified volume plethysmographic technique. Biomed Sci Instrum. 1985;21:1-3.

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19 Suckert R. Experimental models for traumatic oedemas in rat paws. Med Pharmacol Exp. 1967;17:43-50.

20 Browse NL, Negus D. Prevention of postoperative leg vein thrombosis by electrical muscle stimulation; an evaluation with 125 I-labelled fibrinogen. Br Med J [Clin Res). 1970;3:615-618.

21 Cheng N, Van Hoof H, Bockx E, et al. The effects of electric currents on ATP generation, protein synthesis, and membrane transport in rat skin. Clin Orthop. 1982;171:264-272.

22 Deport PH, Cheng N, Hoogmaxtens MJ, et al. The effect of pulsed electromagnetic fields on protein synthesis and membrane transport in rat skin. Trans Bioelec Repair and Growth Soc. 1982;2:34.

23 Bourguignon GJ, Bourguignon LYW. Electrical stimulation of protein and DNA synthesis in human fibroblasts. FASEBJ 1987;1:398-402.

24 Arvidsson I, Eriksson E. Postoperative TNS pain relief after knee surgery: an attempt to objective evaluation. Orthopedics. 1986;9:13461351.

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K Cosgrove, PT, is Staff Physical Therapist, Good Samaritan Hospital, 5601 Loch Raven Blvd, Baltimore, MD 21239. Address all correspondence to Ms Cosgrove at 1975 Greenberry Rd, Baltimore, MD 21209 (USA).

G Alon, PhD, PT, is Associate Professor, Department of Physical Therapy, School of Medicine, University of Maryland, 32 S Greene St, Baltimore, MD 21201.

SF Bell, PT, is Staff Physical Therapist, Francis Scott Key Medical Center, 4940 Eastern Ave, B-1 North, Baltimore, MD 21224.

SR Fischer, PT, is Staff Physical Therapist, Magee Rehabilitation Hospital, 6 Franklin Plaza, Philadelphia, PA 19102.

NR Fowler, PT, is Staff Physical Therapist, Good Samaritan Hospital. N Jones, PT, is Staff Physical Therapist, Mills Memorial Hospital, 100 S San Mateo Dr, San Mateo, CA 94401.

JC Myaing, PT, is Part-time Staff Physical Therapist, Holy Cross Hospital, 1500 Forest Glen Rd, Silver Spring, MD 20910.

TM Crouse, PT, is Staff Physical Therapist, Valley Physical Therapy Group, 1306 West Ave, Lancaster, CA 93534.

LJ Seaman, PT, is Staff Physical Therapist, Shady Grove Adventist Hospital, 9901 Medical Center Dr, Rockville, MD 20850.

All authors, with the exception of Dr Alon, were students in the Department of Physical Therapy, School of Medicine, University of Maryland at Baltimore, when this study was completed in partial fulfillment of their bachelor of science degrees.

This study was approved by the University of Maryland institutional Review Board. This article was submitted January 8, 1991, and was accepted September 9, 1991.

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