Influence of high voltage pulsed current on edema formation following impact injury in rats.

In a recent series of studies, Bettany et al[1-3] and Taylor et al[4] demonstrated that cathodal high voltage pulsed current (HVPC) at 120 pulses per second (pps) and voltages 10% less than those needed to evoke visible muscle contractions significantly curbed edema formation in bullfrogs (Rana catesbeiana) following injury caused by impact or hyperflexion. These studies were the first to clearly demonstrate a significant treatment effect for any form of electrical stimulation in inhibiting acute edema. Subsequent studies in our laboratory using bullfrogs as models indicated that (1) anodal HVPC at 120 pps and voltages 10% less than visible motor threshold had no treatment effect,[5] (2) cathodal HVPC at 1 pps and intensities sufficient to produce minimal joint movement (ie, evoke a "muscle pump") failed to curb posttraumatic edema,[6] and (3) cathodal low voltage pulsed current (LVPC) with 620- to 630-microsecond pulse durations and intensities 10% less than visible motor threshold failed to significantly curb edema formation.[7]

No clear understanding of mechanisms underlying treatment effects for edema emerged from these studies, but we did conclude that (1) strong sensory stimulation (ie, 10% less than visible motor threshold) does not by itself produce a treatment effect, (2) strong sensory stimulation and muscle activity evoked by cathodal HVPC at 1 pps does not produce a treatment effect in the early hours after injury, and (3) polarity seems to be important in achieving a treatment effect using HVPC. Because some forms of sensory stimulation and low-level muscle activation fail to inhibit edema formation and because stimulus characteristics we have used are unlikely to depolarize a substantial proportion of small-diameter myelinated or unmyelinated fibers (eg, autonomic and nociceptive fibers), we speculate that whatever HVPC is doing to curb edema is not neuronally based.

Speculations on mechanisms aside, we believe that these findings provide adequate bases for clinical trials of efficacy of cathodal HVPC in controlling acute edema. Bullfrogs, vertebrates that have long been used in investigations of neurophysiology and wound healing, are easy to maintain under anesthesia for prolonged periods without fear of desiccation or having to provide intravenous nutrients. Anesthesia is necessary to eliminate confounding variables such as pain, stress, and movement, which invoke muscle pump (see Fish et al[5] for a more thorough discussion of the rationale for the frog model). Although our experiments on frogs have all been conducted at room temperature (near the high end of the normal range of temperatures for free-ranging bullfrogs) and we traumatized equally both limbs of each animal and statistically compared treated limbs with untreated limbs, bullfrogs are poikilotherms (cold-blooded) and may therefore respond differently from homeothermic (warm-blooded) mammals to trauma and to treatment. To determine whether this is true, and to test our assumption that the bullfrog model can serve to predict therapeutic outcomes in mammals, we elected to replicate on rats our initial study of the effects of HVPC on edema formation following impact injury in bullfrogs.[1]

Method

Subjects

Eighteen Zucker-lean rats and 6 Sprague-Dawley rats, weighing 300 to 593 g, were used in this study. The Zucker rats were retired breeders of both sexes. These were large rats, but they had relatively small feet. The Sprague-Dawley rats, of both sexes, were younger and smaller than the Zucker rats and had relatively large feet. The method of anesthesia and handling procedures, including mode of traumatizing hind limbs and sacrifice, were approved by the Institutional Laboratory Animal Care Committee of the State University of New York at Buffalo.

Instrumentation

Trauma was induced by a procedure modified after the technique described by Bettany et al.[1] This procedure consisted of dropping a steel rod weighing 85.5 g through a vertical tube from a height of 30 cm. A rectangular piece of plastic (2 x 2 x 0.5 cm) was interposed between the hind limb and the tube to distribute force of impact and prevent rupture of skin. Limb volumes were determined by a water displacement apparatus consisting of an immersion vessel and overflow chamber connected by rubber tubing. This was the same apparatus used for frog studies,[1-3] except that the immersion vessel used in this study was smaller to accurately measure the smaller feet of rats. Animals, suspended in cloth slings, were lowered by a camera boom until the hind limb to be measured was submerged to a line painted on the leg. Water that subsequently spilled from the overflow chamber was collected in a 100-mL beaker and weighed on a microbalance(*1) to determine limb volume. Reliability of this volume measurement system was determined by performing 20 volume measurements of each hind limb of a rat. Estimation of reliability using one-way fixed-effects analysis of variance (ANOVA) procedures yielded an intraclass correlation coefficient of .99.

High voltage pulsed current was supplied by identical Intelect 500S stimulators.[unkeyable] Output consisted of twinspiked monophasic pulsed current. Spikes, of 5 and 8 microseconds duration (at 50% of peak voltage), were separated by an interphase interval of 75 microseconds. These pulses were delivered continuously at 120 pps. Cloth slings used to suspend the animals were lined with 9 x 3.6-cm carbon-rubber electrodes[unkeyable] (one per sling), which functioned as anodes; these electrodes were coated with electrode gel and applied to the animals' shaved abdominal walls. In most of our previous studies, dispersive electrodes were placed on the animals' hips, rather than under the animals' abdomens, as we did in this study. Consequently, dispersive electrodes were larger relative to active electrodes in this study, and the distance between electrodes was slightly greater. Current flow, however, was still proximal to distal through the site of injury. Carbon-rubber electrodes[unkeyable] were immersed in beakers, one per beaker and one beaker for each hind limb, so that water in the beakers served as cathodes.

Procedure

In addition to the primary study to determine the effects of cathodal HVPC on edema, we conducted a small-scale investigation of edema caused by trauma relative to that caused by dependent positioning of limbs.

Table 1.  Analysis of Variance Summary for Repeated Measures for
Trauma Effect
Source                  df   SS      MS      F       P(a)
Trauma (A)              1    32.06   32.06   11.06   .015
Repeated measures (B)   8     8.70    1.09   30.59   .0001
AB                      8     1.80    0.22    6.31   .0001
(a) P<.05.

Each rat was anesthetized by intraperitoneal injection of Inactin[unkeyable] (5-ethyl-5[1-methyl-propyl]-2-thiobarbituate) (1 g per kilogram of body weight). Inactin is a new anesthetic that is not yet commercially available in the United States. This drug permitted us to anesthetize rats for more than 4 hours with a single dose, which minimized stress and discomfort to the animals and allowed us to complete our experiment with minimal handling of our subjects. That is, limbs were not reoriented or manipulated, because repeated injections were seldom necessary. On occasion, however, rapid respiration or presence of corneal reflexes or muscle twitches indicated the need for additional anesthesia, which was provided in 0.1-mL doses. Lines were painted just proximal to malleoli prior to initial limb-volume measurements. For the pilot study, one hind limb of each of four Sprague-Dawley rats was randomly selected for trauma. These four limbs and each hind limb of all rats in the primary study were injured by dropping a steel rod onto the plantar aspect of each foot just distal to malleoli.

Both hind limbs of the rats in the pilot study were measured every half hour for 4 hours beginning immediately after trauma was induced. For the other rats, a series of four 30-minute HVPC treatments was begun within 5 minutes after injury; a 30-minute rest period followed each treatment. Each treatment began with the rat's hind limbs immersed in separate 1-L beakers. Voltage was then slowly increased until minimal limb movement was observed in both limbs (ie, motor threshold was determined). One limb, randomly selected, then received cathodal 120-pps HVPC at 90% of visible motor threshold (between 20 and 50 V). The other limb, having been briefly brought to motor threshold, received no further stimulation and thus served as a control. The brief contraction was induced in the control limb to match the contraction induced in the treated limb, thereby minimizing the possibility of differential muscle-pump effects. Animals were suspended in slings with their hind limbs in dependent position throughout data collection.

Volume measurements were obtained before and after trauma and after each treatment and rest period by immersing each hind limb to its premarked line. All data were collected by a rater ignorant of treatment assignment. Skin was incised over trauma sites just before sacrifice of all subjects to confirm that volume changes were attributable to edema formation and not frank bleeding.

Not surprisingly, immobility and dependent positioning produced some edema, but trauma produced significantly more edema (Tab. 1, Fig. 1). These results suggest that the trauma-induced edema we created was readily distinguishable from the "background edema" associated with dependent positioning. The high respiratory rate of the rats, even when slowed by anesthesia, undoubtedly resulted in some dehydration over the 4 hours or more of anesthesia. This dehydration probably reduced the total amount of edema and hence influenced our measurement of limb volumes. This putative reduction in swelling, however, was in some measure compensated by the limbs remaining in dependent position throughout data collection. Because both treated and untreated limbs were handled the same, except during treatment, whatever effects dehydration and dependent positioning may have had should have manifested uniformly and therefore should not have effected any relative differences between limbs. These limitations seemed to us to present fewer difficulties than directly interdicting the vascular systems of our subjects by intravenous administration of anesthetic, the standard means by which long-term anesthetic is delivered.

Data Analysis

Data were expressed as changes from pretrauma hind-limb volumes per kilogram of body weight (ie, data were normalized to minimize effects of size on amount of swelling). Analyses of variance for repeated measures were used to test the null hypotheses that (1) trauma would not cause edema beyond that caused by dependent positioning and (2) treatment would not influence posttraumatic limb volumes. A .05 level of significance was selected. Student's post hoc paired t tests were used to assess differences (P<.05) between treated and untreated limb-volume changes at selected repeated measures.

Results

In the primary study, wherein bilateral trauma but unilateral treatment was applied, volumes of treated limbs were significantly less than volumes of untreated limbs after the second treatment (Tabs. 2 and 3, Fig. 2). That is, cathodal HVPC at 120 pps and intensities 10% below visible motor threshold significantly curbed edema formation in rats.

Discussion

High voltage pulsed current retarded edema formation following impact injury in rats (Tab. 2, Fig. 2). It did so despite limbs being in dependent position throughout treatment and rest periods. This result is consistent with findings in studies of frogs that were injured by either impact[1,3,4] or hyperflexion.[2] A position treatment effect in rats not only corroborates previous findings, but suggests a more general applicability. That is, positive treatment effects in frogs, and now rats, suggest that whatever HVPC does to inhibit edema formation depends on aspects of physiology shared by quite distantly related vertebrates. Indeed, that physiology may be common to many, if not all, vertebrates. If so, it seems reasonable to expect that humans, too, might share that same physiology and therefore be expected to respond to HVPC in a similar fashion. At a minimum, finding a positive treatment effect in frogs and rats forms a compelling rationale for initiating trials of efficacy of HVPC in inhibiting edema formation in humans. Table 2. Analysis of Variance Summary for Repeated Measures for Treatment Effect

Source                  df   SS      MS      F       P(a)
Treatment (A)           1    18.00   18.01   10.41   .0026
Repeated measures (B)   8     6.45    0.81   12.69   .0001
AB                      8     9.14    1.14   17.97   .0001

(a) P<.05.

As we have done in previous studies using bullfrogs, we chose to keep the rats anesthetized and their limbs in dependent position throughout data collection. We believe that maintaining anesthesia reduces stress and possibly sympathetic effects on edema. Certainly, anesthesia minimizes pain, although that may also reduce the total amount of edema produced.[8] Maintaining anesthesia also precludes any exercise effects produced by muscle activity (ie, muscle pump) or behavior such as rubbing or licking that may in some way influence limb volumes.

Anesthesia probably retards physiologic activities, including those germane to inflammation and wound healing, but clearly does not prohibit such activities. Limbs were kept in dependent position throughout treatment and rest periods for ease of treatment (immersion technique) and measurement (also immersion technique), but also to avoid potentially therapeutic positions, which might liberally be interpreted to be any position but dependent. Like some other studies in our series, we imposed a more stringent test of efficacy of HVPC to ensure that positioning was not a factor. Efficacy of HVPC for edema control might be underestimated using the model described in this report because dependent positioning may mask a small, but positive, treatment effect. We report a signficant treatment effect for HVPC in this study despite the additional challenge of dependent positioning. Table 3. Results of Post Hoc Paired t Tests for Posttrauma Measurements (Comparisons of Treated and Untreated Limb-Volume Increases)

                           Mean
Measurement         df     X-Y(a)     Paired t     P
Posttrauma          19     .168       1.207        .2423
Posttreatment 1     19     .157       1.142        .2677
Postrest 1          19     .223       1.564        .1343
Posttreatment 2     19     .437       3.112        .0057(b)

(a) X=change in treated limb volume per kilogram of body weight, Y=change in untreated limb volume per kilogram of body weight. (b) Significant at P<.0125 (using four repeated Bonferroni adjusted t tests, P=.05).

This study did little to enhance our understanding of the mechanism(s) underlying the demonstrated treatment effects of HVPC. We doubt that HVPC produces electrical fields of sufficient strength to cause directional migration of plasma proteins (and their attendant water) because the duty cycle of this form of electrical stimulation is so brief (less than 1%). Whatever field effect HVPC may exert is likely to be reversed, or at least negated, during the comparatively long periods between pulse pairs. We speculated earlier (see introduction and Fish et al[56] that, because the electrical stimulation used in this and previous studies of edema[1-7] was delivered at only 90% of visible motor threshold, thresholds of substantial numbers of smaller or unmyelinated sympathetics were probably not exceeded and therefore were unlikely to account for our results.

Stimulation just below motor threshold using anodal HVPC or LVPC does not uniformly result in the curbing of edema formation.[5,7] Even when low-level motor activities were initiated with HVPC, we were unable to curb edema formation.[6] These observations suggest to us that, whatever sensory-level HVPC is doing to curb edema, the mechanism of action is probably not neuronally mediated. We further speculate, following Reed,[9] that HVPC may be affecting microvessel permeability, which could, in turn, affect both fluid and protein loss to interstitial spaces. Indeed, we believe that the positive treatment effects demonstrated in this and other studies in our series and the lack of treatment effect observed by Mohr et al[10] and Cosgrove et al(11) might be ratinalized, at least in part, by this hypothesis.

Mohr et al[10] and Cosgrove et al,[11] in the only other controlled studies on edema, observed that treatment applied at 24-hour intervals for 20 and 60 minutes, respectively, did not accelerate diminution of already resolving edema. If, as Reed[9] suggests, HVPC inhibits edema by decreasing permeability of microvasculature, then beginning submotor treatment with HVPC after edema is well established may be counterproductive. That is, reducing permeability after plasma proteins (and water) have already escaped the vascular system might be ineffective and may even slow resorption of water into the vasculature. It is possible that our results and those of Mohr et al[10] and Cosgrove et al[11] differ because we applied treatment during the acute phase of injury (when proteins and fluids were still being lost to the interstitium), whereas they applied HVPC after edema was fully formed and resolving on its own (ie, during the subacute phase when plasma proteins were no longer profusely leaking from the vascular system).

Whatever the underlying mechanisms, the results of this and other studies in our series and those of Mohr et al[10] and Cosgrove et al[11] suggest that current clinical protocols for treatment of acute edema need revision. Efficacy of clinical protocols, however, has never been documented,[12] so altering or abandoning them should not be cause for consternation.

We believe that observed treatment effects for HVPC in controlling edema formation in frogs, and now rats, provide a strong rationale for beginning trials of efficacy of cathodal HVPC in inhibiting acute edema formation in humans. If a similar treatment effect is observed in humans, then further trials will be necessary to begin to develop protocols. In the meantime, further studies using non-human models are necessary to determine how electrical stimulation curbs edema formation.

Conclusions

An aggressive regimen of four 30-minute treatments separated by four 30-minute rest periods of continuous 120-pps cathodal HVPC at voltages 10% less than those needed to evoke visible muscle contraction delivered via immersion technique retarded edema formation after impact injuries in rats. This outcome parallels those of repeated studies using identical stimulators and stimulation characteristics, but on frogs. This finding suggests that the mechanism(s) of edema formation affected by HVPC are probably the same in a wide variety of vertebrates, including humans, and that clinical trials of efficacy of HVPC in controlling acute edema are warranted.

Acknowledgments

We thank Dr Harold Burton for review of the manuscript, Dr Robert Hard for assistance with graphics, Chattanooga Corporation for complimentary use of stimulators, and Dr Brian Murray for providing the anesthetic.

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

[unkeyable]Cattanooga Corp, PO Box 4287, Chattanooga, TN 37405.

[unkeyable]Tenzcare, 3M Corp, Bldg 225-5S-01, 3M Center, St Paul, MN 55144-100.

[unkeyable]BYK, Andrew Lockwood & Associates, PO Box 366, Sturtevant, WI 53177.

References

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