Research Report
Influence of High Voltage Pulsed Current on Edema Formation ~ollowing1mpact Injury in Rats
High voltage pulsed cuwent (HVPC) has been shown to be efective in curbing posttraumatic edemaformution in ji-ogs. The purpose of this study was to establish the utiliry of HVPC in controlling edema formation in a mammalian model. Both feet of 20 anesthetized rats were traumatized following initial determination of hind-limb volumes. Four 30-minute cathodal HVPC treatments at 120 pulses per second and 90% of visible motor threshold interspersed with 30-minute rest periods were applied to one randomly selected hind limb of each rat via the immersion technique. The other hind limb served as a control. Limb volumes were measured after each treatment and rest period. All data were expressed as changes from pretrauma limb volumes in milliliters per kilogram of body weight; these data were analyzed by repeated-measures analyses of variance and post hoc paired t tests. Volumes of untreated limbs were signijicantly greater than volumes of treated limbs after the second treatment. Evidence of signijicant treatment effects in frogs, and now rats, provides a compelling rationalefor initiating trials of eficucy oJ"cathoda1HVPC in inhibiting acute edema formution in humans. [Mendel FC, W:vlegalaJ4Fish DR. Influence of high voltage pulsed cuwent on edema formation following impact injury in rats. Phys Ther. 1992;72:668-673.]
Frank C Mendel Juli A Wylegala Dale R Flsh
Key Words: Edema; Electrotherapy, electrical stimulation; Wounds and injuries, rats.
In a recent series of studies, Bettany et all-3 and Taylor et a14 demonstrated that cathodal high voltage pulsed current (INPC) at 120 pulses per
second @ps) and voltages 10% less than those needed to evoke visible muscle contractions significantly curbed edema formation in bullfrogs
FC Mendel, PhD, is Associate Professor, Department of Anatomical Sciences, 317 Farber Hall, State University of New York at Buffalo, BuEalo, NY 14214 (USA). Address correspondence to Dr Mendel. JA Wylegala, FT, is a student in the Physical Therapy and Exercise Science Program, State University of New Yod; at Buffilo. DR Fish, PhD, FT, is Associate Professor, Department of Physical Therapy and Exercise Science, and Director, Electrotherapy Research Iaboratory, State University of New York at Buffalo. This article is adapted from a thesis completed in partial fulfillment of the requirements for Ms Wylegala's hlaster of Science degree in exercise science at the State University of New York at Burno. This study vras approved by the Institutional Iaboratory Animal Care Committee of the State University of New ~ o r at k Buffalo. This study vras supported by a grant from the New York Chapter of the American Physical Therapy Association.
(Ram catesbeiana) following injury caused by impact o r 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? 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
This article ,was submitted September 27, 1990, and was accepted May 15, 1992.
Physical ?'herapy/Volume 72, Number 9fieptember 1992
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668 / 55
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 o r having to pmvide intravenous nutrients. Anesthesia is necessary to eliminate confounding variables such as pain, stress, and movement, which invoke muscle pump (see Fish et als 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-mging b u l h g s ) and we traumatized equally both limbs of each animal and statistically compared treated limbs with untreated limbs, bullfrogs are poikilotherms (cold-blooded) and may there-
fore respond d8erently 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.
Trauma was induced by a procedure modified after the technique described by Bettany et al.l 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 X0.5 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,'-3 except that the immersion vessel used in this study was smaller to accurately mea-
'Mettler Instrument Corp, 29 Nassau St, Princeton, NJ 08520. +ChattanoogaCorp, PO Box 4287, Chattanooga, TN 37405 *~enzcare,3M Corp, Bldg 225-5s-01, 3M Center, St Paul, MN 55144-1000.
56 / 669
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sure 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* 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 fixedeffects analysis of variance (ANOVA) procedures yielded an intraclass correlation coefficient of .99. High voltage pulsed current was supplied by identical Intelect 500s stimulators.+ 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 9x36-cm carbon-rubber electrodes* (one per sling), which functioned as anodes; these electrodes were coated with electrode gel and applied to the mimals' 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* 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.
Physical Therapy /Volume 72, Number 9lSeptember 1992
-
Table 1. Analysis of Variance Summary for Repeated Measures for Trauma Effect Source
df
SS
MS
F
P
Trauma (A)
1
32.06
32.06
11.06
.015 .0001 .0001
Repeated rrleasures (B)
8
8.70
1.09
30.59
AB
8
1.80
0.22
6.31
Each rat was anesthetized by intraperitoneal injection of 1nactin5 (5-ethyl-511-methyl-propyll-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 hor~rswith a single dose, which mir~imizedstress and discomfort to the animals and allowed us to complete our experiment with minimal handling of our subjects. That is, limbs were not reoriented o r manipulated, because repeated injections were seldom necessary. On occasion, however, rapid respiration o r presence of corneal reflexes o r 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.
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 HVF'C 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 ditferential muscle-pump effects. Animals were suspended in slings with their hind limbs in dependent position throughout data collection.
Both hind limbs of the rats in the pilot study were measured every half hour for 4 hours begiming immediately after trauma was induced. For the other rats, a series of four 30-minute HVF'C 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
Not surprisingly, immobility and dependent positioning produced some edema, but trauma produced significantly more edema (Tab. 1, Fig. 1). These results suggest that the traumainduced edema we created was readily distinguishable from the "background edema" associated with dependent positioning. The high respiratory rate of the rats, even when
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.
#BYK, Andrew Lockwood & Associates, PO Box 366, Stunevant, WI 53177.
Physical 'I'herapy/Volume 72, Number 9beptember 1992
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slowed by anesthesia, undoubtedly resulted in some dehydration over the 4 hours o r 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 HVF'C at 120 pps and intensities 10% below visible motor threshold significantly curbed edema formation in rats.
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.
3 h
m
5E
Y
a,
m
2
E
a
d g=I $a
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) o r behavior such as rubbing or licking that may in some way influence limb volumes.
U Traumatized Untraumatized
I
1
E 3 0
1
0
3
2
4
HOIJrs Posttrauma Flgure 1. Mean changes in traumatized and untraumatized rat hind-limb volumes over time. Vertical lines depict standard errors. TheeJirstpair of means are immediate posttrauma values; thereafter, pairs of means represent data collected after each 30-minute treatment and i n t e r n i n g 30-minute rest periods.
Discussion
findings in studies of frogs that were injured by either impact18384 o r hyperf l e ~ i o nA. ~positive 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
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
Table 2. Analysis of Variance Summaty for Repeated Measures for Treatment Effect Source
F
Pa
18.01
10.41
,0026
0.81
12.69
.0001
1.14
17.97
.0001
df
SS
MS
Treatment (A)
1
18.00
Repeated measures (B)
8
6.45
AB
8
9.14
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i
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 significant treatment effect for HVPC in this study despite the additional challenge of dependent positioning.
Physical Therapy/Volume 72, Number 9/September 1992
,
-
Table 3. Results of Post Hoc Paired t Testsfor Posttrauma Measurements (Compa~sonsof Treated and Untreated Limb-Volume Increases)
Measurement
df
Po~ttraum~a
19
Posttreatrr~ent1
19
Postrest 1
19
Posttreatment 2
19
Mean X-Ye
"X=changc: in treated limb volume per kilogram of body weight, Y=change in untreated limb volume per kilogram of body weight. 'significant at P c . 0 1 2 5 (using four repeated Bonferroni adjusted r 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, o r at least negated, during the comparatively long periods between pulse pairs. We speculated earlier (see introduction and Fish et al5) that, because the electrical stimulation used in this and previous studies of edema'-' was delivered at only 90% of visible motor threshold, thresholds of substantial numbers of smaller o r unmyelinated syrnpathetics 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.517Even when lowlevel motor activities were initiated with HVPC, we were unable to curb edema formation.6 These observations suggest to us that, whatever sensorylevel HVPC is doing to curb edema, the mechanism of action is probably not neuronally mediated. We further speculate, following Reed? 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 all0 and Cosgrove et al" might be rationalized, at least in pan, by this hypothesis.
I
Hours Posttrauma
Flgure 2. Mean changes in treated and untreated rat hind-limb volumes over time. Vertical lines depict standard errors; asterisks denote absence of signijicant dz$ierences. Means ojrtreated and untreated limb volumes are signiJcantly diferent after the second treatment (at 1.5 hours posttrauma). Thef i t pair of means are immediate posttrauma values; thereafter,pairs of means represent data collected after each 30-minute treatment and intervening 30-minute rest p e n e n d . Physical Therapy/Volume 72, Number 9/September 1992
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Mohr et all0 and Cosgrove et al," 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 Reed9 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 all0 and Cosgrove et all1 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 lealung from the vascular system). Whatever the underlying mechanisms, the results of this and other studies in our series and those of Mohr et all0 and Cosgrove et all1 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.
then further trials will be necessary to begin to develop protocols. In the meantime, further studies using nonhuman 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 aEected 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 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,
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.
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References 1 Bettany JA, Fish DR, Mendel FC. Influence of high voltage pulsed direct current on edema formation following impact injury. Phys T h e 1990;70:219-224. 2 Bettany JA, Fish DR, Mendel FC. Effect of high voltage pulsed direct current on edema formation following hyperflexion injury. Arch Phys Med Rehabil. 1990;71:677481. 3 Bettany JA, Fish DR, Mendel FC. Influence of cathodal high voltage pulsed current on acute edema. J Clin Electrophysiol. 1990;2:5-8. 4 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;72:6348. 5 Fish DR, Mendel FC, Schultz AM, GottsteinYerke LM. Effect of anodal high voltage pulsed current on edema formation in frog hind limbs. Phys Ther 1991;71:724733. 6 Taylor K, Fish DR, Mendel FM, Bunon HW. Effect of electrically induced muscle contractions on posttraumatic edema formation in hog hind limbs. Phys Ther. 1992;72:127-132. 7 Kames JL, Mendel FC, Fish DR. Effects of low voltage pulsed current on edema formation in frog hind limbs following impact injury. Phys Ther. 1992;72:273-278. 8 Fields HL. Pain. New York, NY:McGraw-Hill Inc; 1987. 9 Reed BV. Effect of high voltage pulsed electrical stimulation on microvascular permeability to plaqma proteins: a possible mechanism in minimizing edema. Phys Ther. 1988;68: 491-495. 10 Mohr TM, Akers TK,Iandry RG. Effect of high voltage stimulation on edema reduction in the rat hind paw. Phys Thm. 1987;67: 1703-1707. 1 1 Cosgrove KA,Alon G, Bell SF, et al. The electrical effect of two commonly used clinical stimulators on traumatic edema in rats. Phys Ther. 1992;72:227-233, 12 Mendel FC, Fish DR. New perspectives in edema control via electrical stimulation. Journal of Athletic Training. In review.
Physical Therapy /Volume 72, Number 9lSeptember 1992
Influence of High Voltage Pulsed Current on Edema Formation ~ollowing1mpact Injury in Rats
High voltage pulsed cuwent (HVPC) has been shown to be efective in curbing posttraumatic edemaformution in ji-ogs. The purpose of this study was to establish the utiliry of HVPC in controlling edema formation in a mammalian model. Both feet of 20 anesthetized rats were traumatized following initial determination of hind-limb volumes. Four 30-minute cathodal HVPC treatments at 120 pulses per second and 90% of visible motor threshold interspersed with 30-minute rest periods were applied to one randomly selected hind limb of each rat via the immersion technique. The other hind limb served as a control. Limb volumes were measured after each treatment and rest period. All data were expressed as changes from pretrauma limb volumes in milliliters per kilogram of body weight; these data were analyzed by repeated-measures analyses of variance and post hoc paired t tests. Volumes of untreated limbs were signijicantly greater than volumes of treated limbs after the second treatment. Evidence of signijicant treatment effects in frogs, and now rats, provides a compelling rationalefor initiating trials of eficucy oJ"cathoda1HVPC in inhibiting acute edema formution in humans. [Mendel FC, W:vlegalaJ4Fish DR. Influence of high voltage pulsed cuwent on edema formation following impact injury in rats. Phys Ther. 1992;72:668-673.]
Frank C Mendel Juli A Wylegala Dale R Flsh
Key Words: Edema; Electrotherapy, electrical stimulation; Wounds and injuries, rats.
In a recent series of studies, Bettany et all-3 and Taylor et a14 demonstrated that cathodal high voltage pulsed current (INPC) at 120 pulses per
second @ps) and voltages 10% less than those needed to evoke visible muscle contractions significantly curbed edema formation in bullfrogs
FC Mendel, PhD, is Associate Professor, Department of Anatomical Sciences, 317 Farber Hall, State University of New York at Buffalo, BuEalo, NY 14214 (USA). Address correspondence to Dr Mendel. JA Wylegala, FT, is a student in the Physical Therapy and Exercise Science Program, State University of New Yod; at Buffilo. DR Fish, PhD, FT, is Associate Professor, Department of Physical Therapy and Exercise Science, and Director, Electrotherapy Research Iaboratory, State University of New York at Buffalo. This article is adapted from a thesis completed in partial fulfillment of the requirements for Ms Wylegala's hlaster of Science degree in exercise science at the State University of New York at Burno. This study vras approved by the Institutional Iaboratory Animal Care Committee of the State University of New ~ o r at k Buffalo. This study vras supported by a grant from the New York Chapter of the American Physical Therapy Association.
(Ram catesbeiana) following injury caused by impact o r 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? 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
This article ,was submitted September 27, 1990, and was accepted May 15, 1992.
Physical ?'herapy/Volume 72, Number 9fieptember 1992
Downloaded from https://academic.oup.com/ptj/article-abstract/72/9/668/2728986 by Washington University in St. Louis user on 08 June 2018
668 / 55
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 o r having to pmvide intravenous nutrients. Anesthesia is necessary to eliminate confounding variables such as pain, stress, and movement, which invoke muscle pump (see Fish et als 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-mging b u l h g s ) and we traumatized equally both limbs of each animal and statistically compared treated limbs with untreated limbs, bullfrogs are poikilotherms (cold-blooded) and may there-
fore respond d8erently 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.
Trauma was induced by a procedure modified after the technique described by Bettany et al.l 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 X0.5 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,'-3 except that the immersion vessel used in this study was smaller to accurately mea-
'Mettler Instrument Corp, 29 Nassau St, Princeton, NJ 08520. +ChattanoogaCorp, PO Box 4287, Chattanooga, TN 37405 *~enzcare,3M Corp, Bldg 225-5s-01, 3M Center, St Paul, MN 55144-1000.
56 / 669
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sure 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* 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 fixedeffects analysis of variance (ANOVA) procedures yielded an intraclass correlation coefficient of .99. High voltage pulsed current was supplied by identical Intelect 500s stimulators.+ 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 9x36-cm carbon-rubber electrodes* (one per sling), which functioned as anodes; these electrodes were coated with electrode gel and applied to the mimals' 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* 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.
Physical Therapy /Volume 72, Number 9lSeptember 1992
-
Table 1. Analysis of Variance Summary for Repeated Measures for Trauma Effect Source
df
SS
MS
F
P
Trauma (A)
1
32.06
32.06
11.06
.015 .0001 .0001
Repeated rrleasures (B)
8
8.70
1.09
30.59
AB
8
1.80
0.22
6.31
Each rat was anesthetized by intraperitoneal injection of 1nactin5 (5-ethyl-511-methyl-propyll-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 hor~rswith a single dose, which mir~imizedstress and discomfort to the animals and allowed us to complete our experiment with minimal handling of our subjects. That is, limbs were not reoriented o r manipulated, because repeated injections were seldom necessary. On occasion, however, rapid respiration o r presence of corneal reflexes o r 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.
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 HVF'C 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 ditferential muscle-pump effects. Animals were suspended in slings with their hind limbs in dependent position throughout data collection.
Both hind limbs of the rats in the pilot study were measured every half hour for 4 hours begiming immediately after trauma was induced. For the other rats, a series of four 30-minute HVF'C 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
Not surprisingly, immobility and dependent positioning produced some edema, but trauma produced significantly more edema (Tab. 1, Fig. 1). These results suggest that the traumainduced edema we created was readily distinguishable from the "background edema" associated with dependent positioning. The high respiratory rate of the rats, even when
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.
#BYK, Andrew Lockwood & Associates, PO Box 366, Stunevant, WI 53177.
Physical 'I'herapy/Volume 72, Number 9beptember 1992
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slowed by anesthesia, undoubtedly resulted in some dehydration over the 4 hours o r 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 HVF'C at 120 pps and intensities 10% below visible motor threshold significantly curbed edema formation in rats.
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.
3 h
m
5E
Y
a,
m
2
E
a
d g=I $a
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) o r behavior such as rubbing or licking that may in some way influence limb volumes.
U Traumatized Untraumatized
I
1
E 3 0
1
0
3
2
4
HOIJrs Posttrauma Flgure 1. Mean changes in traumatized and untraumatized rat hind-limb volumes over time. Vertical lines depict standard errors. TheeJirstpair of means are immediate posttrauma values; thereafter, pairs of means represent data collected after each 30-minute treatment and i n t e r n i n g 30-minute rest periods.
Discussion
findings in studies of frogs that were injured by either impact18384 o r hyperf l e ~ i o nA. ~positive 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
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
Table 2. Analysis of Variance Summaty for Repeated Measures for Treatment Effect Source
F
Pa
18.01
10.41
,0026
0.81
12.69
.0001
1.14
17.97
.0001
df
SS
MS
Treatment (A)
1
18.00
Repeated measures (B)
8
6.45
AB
8
9.14
58 / 671
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i
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 significant treatment effect for HVPC in this study despite the additional challenge of dependent positioning.
Physical Therapy/Volume 72, Number 9/September 1992
,
-
Table 3. Results of Post Hoc Paired t Testsfor Posttrauma Measurements (Compa~sonsof Treated and Untreated Limb-Volume Increases)
Measurement
df
Po~ttraum~a
19
Posttreatrr~ent1
19
Postrest 1
19
Posttreatment 2
19
Mean X-Ye
"X=changc: in treated limb volume per kilogram of body weight, Y=change in untreated limb volume per kilogram of body weight. 'significant at P c . 0 1 2 5 (using four repeated Bonferroni adjusted r 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, o r at least negated, during the comparatively long periods between pulse pairs. We speculated earlier (see introduction and Fish et al5) that, because the electrical stimulation used in this and previous studies of edema'-' was delivered at only 90% of visible motor threshold, thresholds of substantial numbers of smaller o r unmyelinated syrnpathetics 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.517Even when lowlevel motor activities were initiated with HVPC, we were unable to curb edema formation.6 These observations suggest to us that, whatever sensorylevel HVPC is doing to curb edema, the mechanism of action is probably not neuronally mediated. We further speculate, following Reed? 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 all0 and Cosgrove et al" might be rationalized, at least in pan, by this hypothesis.
I
Hours Posttrauma
Flgure 2. Mean changes in treated and untreated rat hind-limb volumes over time. Vertical lines depict standard errors; asterisks denote absence of signijicant dz$ierences. Means ojrtreated and untreated limb volumes are signiJcantly diferent after the second treatment (at 1.5 hours posttrauma). Thef i t pair of means are immediate posttrauma values; thereafter,pairs of means represent data collected after each 30-minute treatment and intervening 30-minute rest p e n e n d . Physical Therapy/Volume 72, Number 9/September 1992
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Mohr et all0 and Cosgrove et al," 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 Reed9 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 all0 and Cosgrove et all1 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 lealung from the vascular system). Whatever the underlying mechanisms, the results of this and other studies in our series and those of Mohr et all0 and Cosgrove et all1 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.
then further trials will be necessary to begin to develop protocols. In the meantime, further studies using nonhuman 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 aEected 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 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,
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.
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References 1 Bettany JA, Fish DR, Mendel FC. Influence of high voltage pulsed direct current on edema formation following impact injury. Phys T h e 1990;70:219-224. 2 Bettany JA, Fish DR, Mendel FC. Effect of high voltage pulsed direct current on edema formation following hyperflexion injury. Arch Phys Med Rehabil. 1990;71:677481. 3 Bettany JA, Fish DR, Mendel FC. Influence of cathodal high voltage pulsed current on acute edema. J Clin Electrophysiol. 1990;2:5-8. 4 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;72:6348. 5 Fish DR, Mendel FC, Schultz AM, GottsteinYerke LM. Effect of anodal high voltage pulsed current on edema formation in frog hind limbs. Phys Ther 1991;71:724733. 6 Taylor K, Fish DR, Mendel FM, Bunon HW. Effect of electrically induced muscle contractions on posttraumatic edema formation in hog hind limbs. Phys Ther. 1992;72:127-132. 7 Kames JL, Mendel FC, Fish DR. Effects of low voltage pulsed current on edema formation in frog hind limbs following impact injury. Phys Ther. 1992;72:273-278. 8 Fields HL. Pain. New York, NY:McGraw-Hill Inc; 1987. 9 Reed BV. Effect of high voltage pulsed electrical stimulation on microvascular permeability to plaqma proteins: a possible mechanism in minimizing edema. Phys Ther. 1988;68: 491-495. 10 Mohr TM, Akers TK,Iandry RG. Effect of high voltage stimulation on edema reduction in the rat hind paw. Phys Thm. 1987;67: 1703-1707. 1 1 Cosgrove KA,Alon G, Bell SF, et al. The electrical effect of two commonly used clinical stimulators on traumatic edema in rats. Phys Ther. 1992;72:227-233, 12 Mendel FC, Fish DR. New perspectives in edema control via electrical stimulation. Journal of Athletic Training. In review.
Physical Therapy /Volume 72, Number 9lSeptember 1992
