Effects of electrical stimulation on lymphatic flow and limb volume in the rat.

Subcontraction high-voltage stimulation SC-HVS) (ie, electrical stimulation below the level needed to elicit a visible contraction) has been widely used in clinical settings for the purpose of reducing edema. Edema reduction is important because edema results in poor oxygenation of the affected tissue,[1] an impaired ability to function, and a decreased healing rate.[2] Acute edema results from tissue trauma or infection as a result of the inflammatory process.

Acute edema occurs as part of the inflammatory response as chemical mediators lead to endothelial leakage of plasma proteins, primarily albumin, and increased capillary hydrostatic pressure. The resulting increased capillary hydrostatic and interstitial oncotic forces draw fluid from the blood vessels into the interstitium.[3] Because albumin is too large to be reabsorbed by blood vessels, it is returned to the bloodstream from the interstitium almost exclusively by the lymphatic system.[4-7] Consequently, when albumin is taken up by the lymph channels, fluid follows and edema decreases. The importance of the oncotic mechanism is readily apparent in cases of lymphatic obstruction, in which protein uptake is inhibited and edema occurs.[4]

The use of SC-HVS could theoretically affect edema either by preventing edema formation[8-10] or by reducing edema that has already been produced.[11] The first hypothesis is that SC-HVS restricts leakage from the microvasculature by decreasing permeability to proteins.[8] This mechanism addresses the prevention of edema and is supported by the research of Reed[8] and Bettany et al.[9] Reed8 found reduced leakage of flourescein-labeled dextran from the microvasculature of the golden hamster cheek pouch receiving high-voltage stimulation as compared with control animals. Bettany et al[9] found that following trauma, formation of edema was significantly less in frogs' limbs immersed in water and treated with SC-HVS (four 30-minute treatments at 1.5-hour intervals of continuous twin pulses at 75 microseconds, 120 Hz, with negative polarity) as compared with the control limbs. Experimental limb volume also continued to be less than that of the control limb throughout 17 hours of subsequent treatment and measurement intervals. Because there was no further reduction of edema with the subsequent SC-HVS treatments, this result cannot be differentiated from prevention of edema versus reduction of edema.

In a follow-up study, Taylor et al,[10] using variables similar to those described (except with a single 30-minute minute treatment), found a reduction in edema for 4.5 hours following treatment. No difference between the control and the experimental limbs was measured during subsequent time periods.

The second hypothesis addresses the reduction of edema. Alon and De Domenico[11] hypothesized that SC-HVS enhances the movement of charged proteins into the lymphatic channels. When an electric field is introduced into an area, charged proteins are put into motion and migration into the lymphatic channels is accelerated. The lymphatic channels' osmotic pressure is thereby increased, hastening the absorption of fluid from the interstitial space.

A study on reduction of edema yielded results that are seemingly contradictory to Alon and De Dominico's theory.[11] Mohr et al[12] applied one daily 20-minute treatment of SC-HVS (continuous mode, pulse rate=80 pulses per second, average current flow=35 [mu]A with the positive electrode placed over the right hip and the negative electrode over the right edematous hind paws of 20 rats) on 3 consecutive days after traumatization by weight drop (50 g, 0.5 cm in diameter, from a height of 50 cm). Volumes of the traumatized limbs were assessed by a plethysmograph. Both the control and experimental groups revealed a volume reduction over the testing days. The reduction of edema in the experimental group, however, did not reach statistically significant levels as compared with the control group animals. A possible flaw in the study by Mohr et al was their failure to establish reliability for their volume measurements.

No studies have been found that examined the effects of SC-HVS on protein uptake and lymphatic flow. In view of the scarcity of research on this subject, the goal of our experiment was to determine whether SC-HVS alters the flow of interstitial substances into the lymphatic system, thereby affecting edema. The twofold purpose was to determine whether SC-HVS applied to the edematous right hind limbs of rats would (1) modify the rate of lymphatic uptake of albumin labeled with Evans blue dye (AL-EBD) and (2) influence limb volume.

Method

Subjects

A total of 30 male Sprague-Dawley rats (mean weight=263 g, SD=48 g) were housed in a light/dark (12 hours each) and temperature-controlled (72 [degrees] F) environment. They were provided food and water ad libitum.

Procedure

The animals were divided into two groups: a control group (n = 15) and an experimental group (n = 15). All animals were initially anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg per kilogram of body weight) and were given subsequent doses as needed. Sodium pentobarbital has been shown not to influence the contraction rate of lymph vessels.[13] Initial paw volume measurements and blood samples were taken from each rat. All of the animals' right hind paws were then injected with AL-EBD, utilizing sterile techniques, between the second and third metatarsals, which created the experimentally induced edema. Over the course of the experiment, the AL-EBD was absorbed from the interstitium into the lymphatics, entered the bloodstream via the thoracic duct, and was monitored through blood samples. Postinjection blood samples and paw volumes were taken. The experimental group received 1 hour of SC-HVS. The control group received a 1-hour sham treatment. Sham treatment consisted of the same procedure used for the experimental group but without the SC-HVS. Volume measurements and blood samples were then taken from both groups as follows: 1 hour postinjection (corresponding to the termination of the sham or SC-HVS treatment), 3 hours postinjection, 5 hours postinjection, and 7 hours postinjection (Tab. 1).

Table 1. Basic Design of the
Experimental Protocol

                         Blood           Limb
                         Sample          Volume

Anesthetization
Preinjection             XXX(a)           XXX
Injection of
  EBD(b)
Postinjection            XXX              XXX
Treatment - 1 h
  Sham
  SC-HVS(c)
1 h postinjection        XXX             XXX
3 h postinjection        XXX             XXX
5 h postinjection        XXX             XXX
7 h postinjection        XXX             XXX

(a) XXX = point at which blood sample and limb
volume were taken.

(b) EBD = Evans blue dye.

(c) SC-HVS = high-voltage electrical stimulation
that did not elicit a visible contraction.

Volumetric Measurements

Volume measurements of the right hind paws were performed using a small-volume plethysmograph as described by Mohr and Akers,[14] with modifications as described by Cosgrove et al.[15] The modifications included the addition of a biopolar needle electrode,(*) an oscilloscope, and a tripod, which increased the accuracy of the volume measurements. The microsyringe of the plethysmograph was set at zero, and water was added until the fluid level met the tip of the needle electrode. The circuit was thereby closed, as indicated on the oscilloscope. To measure the paw volumes, the lateral malleolus of each animal was marked with waterproof ink. The rat was suspended in a rat restrainer from a rod clamped to a camera tripod. Each rat's paw was dampened prior to being lowered into the immersion vessel of the plethysmograph to the level of the paw's ink mark. The immersion of the limb caused a displacement of fluid, which was drawn of by a calibrated microsyringe until the circuit was broken, as indicated on the oscilloscope. The amount of displaced fluid was taken to be equivalent to the animal's paw volume (in milliliters). The procedure was performed until two measurements were within 0.1 mL of each other. The average of the two immersions was used for statistical analysis. The volumetric measurements were performed by the same experimenter (HAC) throughout the study. The experimenter measured the volume of 10 animals' limbs three times, and intratester reliability was assessed by subjecting the data to an intraclass correlation coefficient (ICC[2,1]) procedure.

Blood Sampling and

Measurement of Evans Blue Dye

A mixture was prepared using Evans blue dye (EBD) and rat plasma (2 mg of EBD per milliliter of heparinized rat plasma). At this concentration, all of the EBD binds to albumin.[16] The concentration of the solution was doubled by centrifuging the mixture through an Amicon Centriflo [TM] membrane cone,([dagger]) which removed one half of the fluid volume. This concentration was found to be necessary in order to monitor the EBD in the plasma by the spectrophotometer. This concentrated solution (0.5 mL per kilogram of body weight) was injected into the rats' paws.

To assess the EBD concentration in the plasma of the rats, 200 [mu]L of blood was collected from the nicked tips of the rats' tails into heparinized capillary tubes.[17] After 5 minutes of centrifugation, the plasma was removed and stored in a refrigerator. For analysis, 100 [mu]L of each plasma sample was diluted with 3 mL of 0.9% saline. The concentration of EBD in the solution (absorbance value) was measured by a DW-2 UV, VIS spectrophotometer([double dagger]) using a wavelength of 620 nm, a band pass of 3 nm, and a cuvette path length of 1 cm.[3] Reliability testing of the apparatus was performed.

Electrical Stimulation

The 1 hour of monophasic pulsed SC-HVS was applied to the experimental group animals, with the positive electrode on the plantar surface and the negative electrode on the shaved dorsal surface of the paw.[15] Self-adhesive electrodes([sections]) measuring 2.3 x 1.5 cm were used and did not overlap each other. The voltage setting was determined for each rat by eliciting a visible contraction of the digital muscles and reducing the intensity until the contraction ceased.[12] The setting was readjusted after 15 minutes to account for accommodation. The stimulator([parallel]) was set on continuous mode at a pulse rate of 100 pulses per second, with an average current of 251.54 [mu]A (SD=96.96) and a voltage of 100.62 V (SD=38.78). The control group animals received 1 hour of sham treatment using the same electrode placement.

Data Analysis

The data collected from this research, the amount of AL-EBD in the plasma (absorbance value) and the limb volume measurements (in milliliters), were evaluated by a two-way analysis of variance for repeated measures with an alpha level of .05. A Student-Neuman-Keuls post hoc test was used to determine where significant results had occurred.

Results

Although the study began with 30 rats, only data from 28 rats (15 control group animals and 13 experimental group animals) were included in the data analysis. One rat died as a result of an overdose from the anesthesia. A second rat's data were discarded due to technical error volume of blood from the tail was inadequate).

Evans Blue Dye Concentration

Main effect differences of the AL-EBD between groups and over time were significant. For this study, however, the interaction effect between groups and time was considered to be the most important comparison (Tab. 2). The following interaction effects were of greatest importance:

Table 2. Statistical Analysis of Evans
Blue Dye in Rat Plasma

                MSE      df      F      P

Groups          125.6    24    10.7    0035(a)
Time periods     25.2   120    40.5   .0000(a)
Interaction      25.2   120     4.4   .0013(a)

(a) P >.05.

1. No differences were found between control and experimental groups in the AL-EBD concentration in the preinjection and postinjection blood samples (ie, before treatment began) (Fig. 1).

2. Increases in the amount of AL-EBD were found in the plasma of the experimental group as compared with the control group at the 1-, 3-, 5-, and 7-hour postinjection blood samplings (Fig. 1).

3. The experimental group animals exhibited increases of AL-EBD from the postinjection (ie, pretreatment) values at 1, 3, 5, and 7 hours postinjection, as opposed to 5 and 7 hours postinjection needed in the control group animals before an increase was noted (Fig. 1). The AL-EBD values remained above the postinjection values for both groups in the subsequent time intervals.

4. Successive increases of the AL-EBD occurred in the experimental group between the time periods of postinjection (ie, pretreatment) and 1 hour postinjection ([4.40 [+ or -] 0.62 to 5.77 [+ or -] 1.28] x [10.sup.-3]) and between 1 and 3 hours postinjection ([5.77 [+ or -] 1.28 to 6.95 [+ or -] 1.52] x [10.sup.-3]) (Fig. 1). In contrast, no increase was seen between successive time periods in the control group animals.

The reliability (r) of the spectrophotmetric readings, as determined by ICC (3, 1), was found to be .99.

Limb Volume

Main effect differences of limb volume between the control and experimental groups were not significant throughout the study. A significant main effect alteration, however, was noted between time intervals with both groups combined (Tab. 3, Fig. 2). The interaction effect revealed no difference between groups and time periods (Tab. 3, Fig. 2). The reliability (r) of the limb volume measurements, as determined by ICC (2,1), was .92.

Table 3. Statistical Analysis of Limb
Volume Measurements

              MSE        df       F        P

Groups        0.1168     25      0.23     .6406
Time periods  5.1163    125    133.25     .0000(a)
Interaction   5.1163    125      1.54     .1802

(a) P >.05.

Discussion

Evans Blue Dye

In both the control and experimental groups, preinjection values of AL-EBD were not significantly different from postinjection values. This finding revealed that the AL-EBD was not inadvertently injected into the bloodstream. This finding was important for establishing the credibility of our methods.

The level of AL-EBD was higher in the experimental group than in the control group at 1, 3, 5, and 7 hours postinjection. The SC-HVS apparently exerted a positive effect on movement of the AL-EBD from the interstitium into the bloodstream. Because the albumin was removed from the interstitial space almost exclusively by the lymphatics and entered the bloodstream via the thoracic duct, the amount of AL-EBD in the blood plasma was directly related to lymphatic uptake from the area.[16,18] In addition, recent studies using radiolabeled albumin support the concept that tagged albumin returns to the bloodstream via the lymphatics.[7,19] If the injected AL-EBD had gone directly into the bloodstream, it would have been detected in the postinjection blood samples. The AL-EBD in the plasma, however, did not increase until at least 1 hour postinjection, showing further support that the lymphatics are responsible for plasma uptake.

Both the control and experimental groups showed an increase in AL-EBD concentration in the plasma over the course of the experiment (Fig. 1). A rise in the AL-EBD as compared with postinjection values, however, occurred at 1 hour postinjection in the experimental group animals, as opposed to 5 hours postinjection needed in the control group animals (Fig. 1). This finding substantiates the efficacy of SC-HVS for rapid removal of the AL-EBD from the interstitial area. Due to the enhancement of lymphatic flow inherent during an edematous situation, the control group eventually did exhibit an increase.[19,20] The continual rise in the subsequent AL-EBD values supports our methodology.

The dramatic increases that were found between two successive time intervals in the experimental group (Fig. 1), as compared with the gradual rise in the control group, are another illustration of the beneficial effect of SC-HVS. In addition, SC-HVS continued to exert its effect for 2 hours poststimulation, for the amount of AL-EBD at the 3-hour measurement was also higher in the experimental group than in the control group (Fig. 1). At the present time, we have no explanation for this phenomenon.

We hypothesize that SC-HVS increases lymphatic uptake of AL-EBD by the following mechanisms: (1) The movement of charged proteins into the lymphatic channels is accentuated, and (2) the contraction of lymphatic smooth muscle is enhanced. Alon and De Domenico's theory,[11] as previously discussed, states that the introduction of an electrical field into an area expedites the movement of charged proteins into the lymphatic channels. Hargens and Zweifach[13] studied the contractility of bovine mesenteric lymphatics and showed that when lymph vessel volume was increased, the contractile rate of lymphatic smooth muscles spontaneously increased. Therefore, the hypothesized mechanism is that the SC-HVS facilitated lymph transport by augmenting the movement of AL-EBD into the rats' lymphatic vessels. The fluid drawn into the vessels by the oncotic force of the AL-EBD distended the lumen of the lymphatic vessel and caused a subsequent increase in the rate of lymphatic contraction.[21]

The third mechanism by which lymphatic flow is thought to increase is that SC-HVS enhances the contraction of lymphatic smooth muscle indirectly via the autonomic nervous system. A previous study[22] demonstrated that under certain conditions (frequency = 48 - 96 Hz, voltage = 50 V, 0.3 milliseconds, seconds' duration), lymphatic nerves and smooth muscle could be directly stimulated to facilitate the contractile rate. These electrical stimulation settings are different from those used in our experiment.

McGeown et al[23] found that stimulation of the sympathetic chain increased lymph flow and lymphatic contraction frequency in sheep hindlimb lymphatics. Transmural stimulation of the bovine mesenteric lymphatics directly excited the intramural nerves of lymphatic smooth muscle.[22] In our study, however, we administered electrical stimulation through the skin, as utilized in physical therapy clinics. Electrical stimulation delivered in this manner cannot directly stimulate lymph smooth muscle and autonomic nervous system fibers without activating skeletal motoneurons.[11] In addition, autonomic nervous sytem fibers that innervate lymph smooth muscle share morphological characteristics with pain fibers (ie, small fiber diameter, unmyelinated, low conduction velocity). If stimulation were to elicit direct excitation of intramural lymphatic nerves, the pain would be intolerable to the subjects and there would be unwanted skeletal muscle contractions. Consequently, a hypothesized mechanism by which AL-EBD uptake was increased in this study is through transcutaneous stimulation of sensory neurons, which may have caused the autonomic nervous system to be stimulated. This stimulation may have led to the release of adrenergic substances, which are excitatory to lymph smooth muscle cells and result in spontaneous propulsion of lymph.[24]

Limb Volume

The enhanced lymphatic drainage, as indicated by the increase in AL-EBD concentration in the plasma, did not lead to any differences in limb volume between the control and experimental groups (Fig. 2). This discrepancy between the increased uptake of AL-EBD without a concurrent reduction in limb volume may be due to the fact that the amounts of AL-EBD taken up were measured with a spectrophotometer, which is approximately 10 times more sensitive than are plethysmographic measurements. In addition, although the 1 hour of SC-HVS enhanced lymphatic flow, the time may not have been sufficient to create a significant volume change. These results agree with those of Mohr et al,[12] who found no difference between control and SC-HVS-treated traumatized rat paws. Our results differed from those of other investigations[8-10] in which SC-HVS was initiated immediately following trauma or histamine injection. As demonstrated by Reed,[8] SC-HVS limits the permeability of capillaries to larger molecules, similar to albumin. In our study, labeled albumin was already in the interstitium prior to the electrical stimulation, which may have led to the difference in results.

The fact that limb volume increased over the course of the experiment in both groups was possibly due to (1) an inadvertent inflammatory reaction to the sterile injection procedure or (2) the oncotic force created by the extremely high concentration of AL-EBD mixture in the interstitial space. If the concentration of tagged albumin had been the same as in plasma (and therefore the oncotic force had been less), the SC-HVS may have led to a reduction in the limb volume values.

Based on the results of this study, we believe that SC-HVS has the potential to reduce edema. A 1-hour treatment, however, may not be sufficient in this model. Because a typical treatment protocol using SC-HVS for edema reduction is approximately 20 to 30 minutes in duration, further clinical research on this modality is essential. Future studies should address (1) creating a situation in which the interstitial concentration of proteins would be the same as in plasma, (2) increasing the time of stimulation, (3) continuing the data collection for a longer time period, and (4) using a more sensitive device for measuring limb volume.

Conclusion

This study demonstrated a physiological mechanism by which SC-HVS may exert its effects on edema, because lymphatic uptake of AL-EBD was greater in the experimental group animals than in the control group animals. A reduction in the rat limb volume was not achieved in either the experimental group or the control group. Although SC-HVS may assist in edema reduction via the lymphatics, further studies are needed to show actual reductions in limb volume.

References

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