Effects of Cool-Water Immersion and High-Voltage Electric Stimulation for 3 Continuous Hours on Acute Edema in Rats

Abstract

Objective:

Cool-water immersion (CWI) at 12.8°C (55°F), cathodal high-voltage pulsed current (CHVPC) at 120 pulses per second and 90% of visible motor threshold, or the combination of CWI and CHVPC, applied 30 minutes on, 30 minutes off for 4 hours, are known to curb edema formation after blunt trauma to the hind limbs of rats. Our purpose was to examine the effects of extending treatment times to 3 continuous hours after blunt trauma to the hind limbs of rats.

Design and Setting:

A randomized, parallel-groups design of 22 subjects was used. Volumes of traumatized limbs, randomly assigned to CWI (n = 7), CHVPC (n = 8), or CWI followed by CHVPC (n = 7) were compared with those of injured but untreated limbs with analysis of variance.

Subjects:

Twenty-two anesthetized Zucker lean rats (mass = 293 ± 27 g).

Measurements:

We measured limb volumes immediately before and after trauma and every 30 minutes over the 4-hour experiment.

Results:

Volumes of treated limbs of all 3 experimental groups were smaller than those of untreated limbs (P < .05). No treatment was more effective than another.

Conclusions:

Exposure to either 3 hours of CWI or CHVPC or to 1 hour of CWI followed by 2 hours of CHVPC effectively curbed edema after blunt injury. These results suggest that these common treatments are effective only during application and hint that application should be maintained throughout the period during which edema is forming.

Cryotherapy and electric stimulation, specifically ice and cathodal high-voltage pulsed current (CHVPC), either alone or together, have long been touted as effective treatments for acute edema.^1–7 Unfortunately, unambiguous supporting evidence from controlled clinical trials is lacking for either of these commonly applied therapies. Cool-water immersion^8,9 (CWI) at 12.8°C (55°F) and CHVPC^8,10–12 at 120 pulses per second (PPS) and 90% of visible motor threshold or the combination of CWI and CHVPC⁸ curbed acute edema in laboratory animals when applied for 30 minutes on, 30 minutes off (rests) for the first 4 hours after injury. An observation common to these studies was that edema formation increased faster during periods when treatments were not applied, that is, during rests.^8,9,12,13 Thus, applying treatment continuously, rather than in interrupted bouts of 30 minutes interspersed with 30-minute rest periods, might prove beneficial. Previous research on laboratory animals suggests that application of cryotherapy for extended periods is not therapeutic and may increase limb volumes.^14–16 The use of high-voltage electric stimulation for extended periods for edema management has not been investigated. Our purpose was to examine the effects of extending treatment to 3 continuous hours on edema formation after blunt trauma to the hind limb of rats.

METHODS

Twenty-two Zucker lean rats (Harlan, Indianapolis, IN) weighing an average of 293 ± 27 g were used in this study. Animals were provided food and water ad libitum until the experiment began. The Institutional Laboratory Animal Care Committee of the State University of New York at Buffalo approved anesthesia and handling procedures, including mode of traumatizing hind limbs and sacrifice.

Instrumentation and Procedures

Impact injury was induced by a procedure similar to that described by Mendel et al.¹² This consisted of dropping a steel rod weighing 85.5 g through a vertical tube from a height of 30 cm onto the plantar aspect of each hind foot just distal to the malleoli. A rectangular piece of plastic (2 × 2 × 0.5 cm) was interposed between the foot and the tube to distribute the force of impact. This method of inducing trauma resulted in changes in limb volume that were attributable to edema formation and not frank bleeding; that is, it caused tissue damage without rupturing major vessels.^8,9,12,13 Furthermore, the skin on these rats is translucent, and we observed no change in color throughout the procedures.

Limb Volume Measurement

We immersed a hind limb and measured the amount of water displaced to determine limb volume. The immersion vessel was 2 cm in diameter and 6 cm long. The inferior end of the vessel was tapered and an additional 6 cm long. A 3-way stopcock was attached so the vessel could be rapidly filled through the tapered end of the vessel. A 23-gauge stainless steel tube, 3 cm long with a 90° bend in the middle, was fixed with epoxy to the inside wall so that 1.5 cm of the tube extended into the immersion vessel. The end of the tube outside the vessel was attached via polyethylene tubing and a 23-gauge needle to a 5-cm³ syringe. Using this tubing complex as a siphon, we brought the water level in the immersion vessel to the same level repeatedly. At the exact level as the tip of the stainless-steel tube in the vessel, a 3-cm piece of 2-0 thread was fixed with white plastic tape to the outside surface. We prepared the animals, which were suspended in cloth slings, for volume measurement by painting lines at the level of their malleoli. Animals were then lowered by motorized booms until the lines painted on their hind limbs lined up with the threads fixed to the immersion vessels (at the level of the tips of the stainless-steel tubes). Displaced water was collected in 5-cm³ syringes by siphoning (Figure 1) and weighed on a microbalance (model S-300D, Fisher Scientific, Pittsburgh, PA). The weight of the fluid collected was equivalent to the animal's limb volume (1 mL = 1 mg). Published work from our laboratory has repeatedly established that the limb–volume-measurement system is reliable and valid within ±1%.^8,9,12,13

Figure 1.

Volumetric measurement system. Reprinted with permission from Dolan et al.⁹

Immersion of traumatized limbs was accomplished by lowering the animal via motorized boom until its hind limbs were immersed to the painted lines in 100-mL beakers of water. Water in the beakers was maintained at 12.8°C (55°F) for animals that received CWI. Water for CHVPC and all control limbs was maintained at room temperature 23°C (75°F). We selected this temperature because Matsen et al¹⁵ reported that it had no therapeutic effect and unpublished work from our laboratory confirmed this finding. The injured but untreated limb of each animal served as the control.

Body Temperature

Because anesthesia can cause body temperature to fall,¹⁷ we regulated body temperature throughout these experiments. A rectal probe was inserted 2–3 cm and connected to a telethermometer (Yellow Springs Instruments, Inc, Yellow Springs, OH), and body temperature was monitored and recorded every 30 minutes throughout the experiment. We regulated body temperature between 35 and 37.5°C by directing a 60-W lamp on an animal, typically throughout an experiment. Temperature in the cool-water beaker was also monitored continuously with a telethermometer and probe. Shaved ice chips were added to beakers to maintain the desired temperature.

Experimental Protocol

Each rat was anesthetized by an intraperitoneal injection of sodium pentobarbital (65 mg/kg of body weight), which was supplemented over the course of the 4-hour experiment as needed with doses of one half of the original dose. Boosters were provided when any motion was observed. Animals in the CWI group needed an average of 1.7 boosters during the experiment (range, 1–3), animals receiving CHVPC needed an average of 1.6 boosters (range, 1–2), and those in the CWI + CHVPC group needed an average of 1.8 boosters (range, 1–2). After the animals were anesthetized, their legs, feet, and abdomens were shaved. Animals were then placed in cloth slings and suspended at 45° (caudal end down), with both hind limbs fully exposed and in dependent position. Lines were painted at the level of the malleoli and rectal probes inserted.

The volume (pretrauma) of each hind limb was determined at least twice and the values averaged to determine the measurement. Both hind limbs of each rat were then injured by dropping a steel rod onto the plantar aspect of each foot just distal to the malleoli. Volumes of both hind limbs were again measured and, within 5 minutes after injury, the limbs were immersed in separate 100-mL beakers. Animals were randomly assigned to 1 of 3 treatment groups. Group 1 received 3 continuous hours of CWI at 12.8°C followed by 1 hour of rest in water maintained at 23°C. Group 2 received 3 continuous hours of CHVPC at 120 PPS and 90% of motor threshold, followed by 1 hour of rest in water maintained at 23°C. Group 3 received 1 hour of CWI, followed by 2 hours of CHVPC, followed by 1 hour of rest in water maintained at 23°C. As in previous work, CHVPC was applied at 120 PPS and 90% of visible motor threshold via the immersion technique. The CHVPC was delivered via an Intelect 500S stimulator (Chattanooga Corp, Chattanooga, TN). Output consisted of twin-spiked, monophasic pulsed current. Spikes of 5 and 8 microseconds were separated by an interpulse interval of 75 microseconds. The cloth sling used to suspend the animals was lined with a 9- × 3.6-cm carbon-rubber electrode, which functioned as an anode; the electrode was coated with electrode gel and applied to the shaved abdominal wall. Carbon-rubber electrodes were immersed in treatment beakers, one per beaker, so that water in the beakers served as cathodes. Animals in the CWI + CHVPC group received the simultaneous application per the techniques described above. Limb volumes of treated limbs and contralateral limbs (controls) were measured every 30 minutes during the 4-hour experiment. Throughout treatments, rests, and measurements, animals remained hanging in slings with their limbs in dependent position. After removal from water beakers and before volume measurement, their feet were dabbed with tissue paper to remove adherent water and to minimize evaporative cooling. At no time were the limbs rubbed or squeezed during drying. At the end of an experiment, animals were sacrificed by exposure to carbon dioxide.

Data Analysis

To minimize the effects of body size, data were expressed as changes from pretrauma hind-limb volumes per kilogram of body weight. We analyzed data by means of a 3 × 2 × 9 ([treatment group: CWI, CHVPC, CWI + CHVPC] × [limb: treated, untreated] × [time: 0, 30, 60, 90, 120, 150, 180, 210, 240 minutes]) analysis of variance using SPSS statistical software (version 10.1; SPSS, Inc, Chicago, IL). Bonferroni-Holm post hoc procedures were used to determine significant comparisons. A P < .05 level of significance was set a priori for all analyses.

RESULTS

Treated limbs (0.245 ± 0.024 mL) were smaller than untreated control limbs (0.477 ± 0.033 mL) for all treatment conditions after 30 minutes and remained so throughout the 4-hour experiment (F_1,19 = 48.24, P = .001; Figure 2). No one treatment (CWI, CHVPC, CWI + CHVPC) was more effective than another in curbing acute edema formation (F_2,19 = 1.024, P = .378; Figure 3). The Table presents the calculated effect size and percentage of swelling between treated and untreated limbs at each time interval from 30 to 240 minutes. Calculations indicated a large effect size for all time periods. Continuous treatment generally maintained limb volumes at 50% or less of control limbs.

Figure 2.

Volume change in treated versus untreated limbs over time. Limbs treated by cold-water immersion, cathodal high-voltage pulsed current, or cold-water immersion followed by cathodal high-voltage pulsed current were smaller than untreated limbs for all time intervals except at time 0 (P .05).

Figure 3.

Volume change by treatment over time. Limbs treated with cold-water immersion (CWI), cathodal high-voltage pulsed current (CHVPC), or cold-water immersion followed by cathodal high-voltage pulsed current (CWI + CHVPC) demonstrated no differences in limb volumes across time among the 3 treatments (P .05).

Effect Size and Percentage Treatment Effect

DISCUSSION

The application of CWI (12.8°C) or submotor CHVPC (120 PPS, 10% less than motor threshold) for 3 continuous hours or CWI for 1 hour followed by 2 hours of submotor CHVPC clearly curbed edema formation after acute impact injuries in rats. No one treatment was better than another. Apparently, we are the first to report that extending treatment times beyond the typical 20 to 30 minutes for CWI and CHVPC and sequencing CWI and CHVPC are effective in curbing acute edema formation. These findings corroborate those from previous trials,^8–13 indicating that exposure to CWI or CHVPC or both is effective in curbing acute edema formation after impact injury.

We did not compare the effects of extended treatment times with multiple 30-minute treatments interrupted by 30-minute rests. Therefore, caution must be used when comparing the results of the separate trials. However, our methods, with the exception of treatment time, are essentially the same as, and in some cases identical with, previous protocols.^8–12,18 One obvious similarity in outcomes between this and our previous studies is the observation that volumes of treated limbs increased more during rest periods, that is, when treatment was not applied. Volumes of treated limbs increased during the final hour of the experiment at a rate similar to that of control limbs. Volumes of treated limbs increased 6% after the first 30 minutes and 10% in the 60 minutes after treatment was terminated at 180 minutes. This increase in limb volume in the absence of treatment is consistent with our observations in other experiments using shorter times for modality applications.^8–12,18

Other researchers using animal models have investigated the effects of cryotherapy for extended treatment times ranging from 1 to 24 hours. McMaster and Liddle¹⁶ concluded that no regimen of cooling positively affected swelling after crush injuries in rabbits. They reported that immersion in 30°C for 1 hour after injury was more effective than immersion in 20°C at 24 hours postinjury. Repeated application of cooling in 3 cycles of 1 hour followed by 1 hour of rest resulted in greater residual swelling in limbs that were treated with 20°C and 30°C water than in control limbs. Matsen et al¹⁵ examined the effects of water immersion ranging from 5°C to 25°C on induced tibial fractures in rabbits. They suggested that temperatures between 5°C and 15°C increased limb volumes by causing thermal injuries. In fact, temperatures below 15°C caused an increase in limb volumes, whereas warmer temperatures had no treatment effect. They speculated that the increase in swelling was caused by damage to the superficial tissue caused by cold-induced ischemia. Meeusen and Lievens¹⁹ suggested that application of cold below 15°C injures superficial lymph vessels, which in turn increases permeability. Seemingly, this study was the first to establish that extended exposure to cool water can be effective in curbing acute edema formation. We speculate that the different outcomes may have resulted from different degrees of injury. Severe crush injuries and fractures cause bleeding (ecchymosis), which contributes to total limb volume. Impact injuries delivered in this study were calibrated to cause edema but not frank bleeding.

We know from experience (unpublished) that the injury inflicted on rats causes their limbs to increase in volume for about 4 hours, after which they slowly begin to decrease in volume.^8–10,12 Taylor et al¹⁸ established that a single 30-minute treatment with CHVPC applied immediately after injury curbs edema formation for a number of hours after application. Applying CHVPC late in the acute inflammatory period would likely result in less effect because the rate at which edema forms decreases as the inflammatory process approaches its zenith. In this study, treatments were applied immediately after injury and maintained for the first three quarters of what is known to be a typical acute inflammatory period for the injury inflicted. Again, the cumulative effects of those treatments held volumes of treated limbs to roughly half those of similarly injured control limbs. If more treatment is better, as this study seems to indicate, then applying treatment throughout the entire acute inflammatory period should be expected to result in even greater curbing of edema than observed here.

No one modality or sequence of modalities applied here was better than another in curbing edema formation. Applying them in sequence, however, may be of some practical benefit. Applying CWI immediately after an injury is inexpensive, quick, easy, and effective in reducing pain, although exposure to 12.8°C can cause temporary discomfort or pain not related to the original injury. Yet maintaining this level of cold via immersion for hours or days is cumbersome at best. Forms of cryotherapy other than immersion, such as ice packs or Cryo Cuffs (Aircast, Inc, Summit, NJ), may provide similar results, although controlled trials have not established their efficacy in acute edema management.²⁰ Exactly how cryotherapy curbs edema remains unknown; however, reduced pain, local metabolism, blood flow, and permeability of microvessels are plausible explanations.^4,21–24 Following CWI with a modality more readily applied and tolerated for long periods (eg, CHVPC) could be beneficial in curbing edema. However, applying CHVPC via immersion, as was done here, renders this modality as impractical as applying CWI. Application of CHVPC via large, conforming surface electrodes and small, portable stimulators might render this modality more practical for long-term use. Although there is some evidence that CHVPC affects permeability of microvessel endothelium,^25,26 we do not know how it works. Because the duty cycle is so short (<1%) and the amplitude so low (10% less than motor threshold), we do not believe that the affected tissues are likely to retain a charge, especially for relatively long periods.

Limitations of this study include the use of an animal model and the method of inducing injury. Extrapolation of human responses from those of rats must be done cautiously. Yet animal models have long been an integral component of medical research used to examine the efficacy of treatments before clinical trials in humans. The injury induced here is similar to a contusion in a human.

If longer treatment is better, as suggested here, and if humans respond as did rats to the treatments applied in this study, then common clinical practice for acute athletic injuries may need revision. We speculate, for example, that applying for 20 to 30 minutes cold (0–15°C), CHVPC, or cold followed by CHVPC, even immediately after an acute sprain or contusion, will have little substantive impact on recovery if the inflammatory response continues for many hours or days. Similarly, we speculate that multiple applications of cold, CHVPC, or some combination for 20 to 30 minutes interspersed with long rest periods (hours or days) will have little substantive impact on recovery.

We conclude that the use of CWI (12.8°C), CHVPC (at 120 PPS and 10% less than motor threshold), or CWI followed by CHVPC, applied for 3 continuous hours, curbs acute edema formation by roughly 50% relative to untreated but similarly injured control limbs of rats. Our findings suggest that these treatments work only during application. Potential implications for clinical application are that treatment should begin as soon after injury as possible and that it should be continuous while edema is still forming.