Transcutaneous electrical nerve stimulation at both high and low frequencies reduces primary hyperalgesia in rats with joint inflammation in a time-dependent manner.

Transcutaneous electrical nerve stimulation (TENS) is a noninvasive treatment for pain. The clinical literature examining the effects of TENS on pain is controversial, with systematic reviews showing a positive effect in people with osteoarthritis pain. (1) However, for other conditions, such as chronic low back pain, systematic reviews of the effects of TENS are inconclusive. (2,3) Clinical studies of TENS have used a variety of outcome measures to assess the effectiveness of TENS; these include serf-report pain rating scales, joint function, analgesic intake, primary hyperalgesia (increased responsiveness to nociceptive stimuli at the site of injury), secondary hyperalgesia (increased responsiveness to nociceptive stimuli outside the site of injury), and various outcome measure questionnaires (for a review, see Sluka and Walsh (4)). It is possible that TENS is effective for some measures of pain or function and ineffective for others.

To address these concerns, our laboratory has tested the effectiveness of TENS in animal models of tissue injury. Injection of kaolin and carrageenan into the knee joint produces an initial acute inflammatory response that is converted to chronic inflammation by 1 week. (5) This inflammatory response is associated with decreased latency to withdrawal of the paw in response to heat, decreased mechanical withdrawal threshold of the paw, and decreased compression withdrawal threshold of the inflamed knee joint. (5,6)

The responses of the paw are considered to be secondary hyperalgesia, an increased response to noxious stimuli outside the site of injury. The response of the inflamed muscle is considered to be primary hyperalgesia, an increased response at the site of injury. This model has been well characterized, showing peripheral sensitization of nociceptors measured as increased sensitivity to joint movement, increased spontaneous activity, and increased responsiveness of previously silent neurons. (7,8) Nociceptive neurons in the spinal cord also become sensitized and show an increased response to cutaneous stimuli, an increased response to joint movement, and increased spontaneous firing. (9,10) This model shows good predictability for the effects of drugs used to treat arthritis, both osteoarthritis and rheumatoid arthritis, and thus is used to assess mechanisms of arthritic pain and effectiveness of treatments for arthritic pain.

Our laboratory previously used the kaolin and carrageenan model of joint inflammation as a model of deep-tissue injury to examine the effects of TENS on a variety of measures of nociception and to examine the mechanisms of action of TENS. Specifically, it was shown that the secondary hyperalgesia of the paw produced by acute joint inflammation and chronic muscle inflammation was completely reversed by both high-frequency TENS and low-frequency TENS at sensory amplitude. (11-13) It was also shown that the primary hyperalgesia produced by carrageenan-induced paw inflammation was only partially reversed by high-frequency TENS and was unaffected by low-frequency TENS. (14)

However, this is a model of cutaneous hyperalgesia, and the results could be different in animals with deep tissue pain. Therefore, we tested the effects of both low-frequency TENS and high-frequency TENS on primary hyperalgesia of the knee joint that had been previously inflamed with kaolin and carrageenan. We further tested the effects when the inflammation was acute and when the inflammation was chronic to determine whether TENS was more effective for acute or chronic inflammatory pain. We hypothesized that both low-frequency TENS and high-frequency TENS would reduce primary hyper algesia of the knee induced by joint inflammation.

Method

Animals were housed within the Animal Care Facility at the University of Iowa with a 12-hour light (7:00 AM-7:00 PM), 12-hour dark (7 PM-7 AM) cycle. They were housed 3 per cage with the same littermates throughout the duration of the testing period.

Induction of Inflammation

Male Sprague-Dawley rats (n=56, 250-350 g) were anesthetized with 4% halothane, and 1 knee joint was injected with 100 [micro]L of a mixture of 3% kaolin and 3% carrageenan. The inflammation is considered acute for the first 24 hours, when there is primarily neutrophil infiltration. By 1 week, the inflammation converts to chronic, as identified histologically by macrophage infiltration. (5) This model is used to mimic arthritic conditions and shows good predictability for drug effects. (15)

Measurement of Compression Withdrawal Threshold of the Knee Joint

The compression withdrawal threshold of the knee joint was measured as previously described. (6,16) Animals were acclimated to the restraining device for 5 minutes 3 times per day for 2 consecutive days. On day 3, while the animal was in the restrainer, the experimenter extended one hind limb, and the knee joint was compressed with the measuring device. Thus, animals were acclimated to the restrainer for 2 days before baseline testing and injection of the knee joint with kaolin and carrageenan. The measuring device consisted of 2 strain gauges attached to the inner arm of a forceps. Compression was stopped when the animal withdrew the limb forcefully or when the animal vocalized. The maximum force applied at withdrawal was recorded as the threshold (in grams) for the knee joint. An average for 3 trials was taken at each time period. The time to peak force for mechanical compression of the knee joint was within 1 second of the time of application of force. For all measurements of the compression withdrawal threshold, the experimenter was unaware of the treatment group. Thus, we used the compression withdrawal threshold of the knee joint to measure primary hyperalgesia.

Application of TENS

Transcutaneous electrical nerve stimulation was applied as previously described to the inflamed knee joint by use of commercially available units (EMPI Eclipse+ *). (11) The TENS protocol outlined below is identical to that shown previously to produce a full reversal of secondary hyperalgesia at 4 hours after the induction of knee joint inflammation with carrageeanan. (11) In this protocol, we modulated the frequency and kept all other parameters (amplitude, pulse duration, and waveform) constant between the 2 frequencies. This strategy allowed a comparison of frequency differences without confounding differences in pulse duration or amplitude (eg, low frequency, high amplitude, and long pulse duration versus high frequency, low amplitude, and short pulse duration).

Briefly, rats were lightly anesthetized with 1% to 2% halothane, the knee joint was shaved, and 2 small pregelled adhesive electrodes (1.27-cm [0.5-in] diameter) were placed on the medial and lateral aspects of the inflamed knee joint. Animals were observed continuously during TENS to ensure adequate anesthesia and to ensure that the electrodes remained in contact with the skin. TENS then was delivered at either a low frequency (4 Hz) or a high frequency (100 Hz); all other parameters were kept constant, as follows: 100-micro-second pulse duration, asymmetrical biphasic square wave, 20-minute duration, and sensory amplitude. Sensory amplitude was determined by increasing the amplitude until a motor contraction was observed and then decreasing the amplitude to just below the motor contraction threshold. Rats that received the sham treatment (sham TENS) were anesthetized with 1% to 2% halothane, the knee joint was shaved, and electrodes were placed on the inflamed joint. Importantly, 3 rats always were anesthetized with the same vaporizer; at least 1 rat receiving the sham TENS treatment and 1 rat receiving the active TENS treatment were anesthetized at the same time. This procedure ensured that there always were animals in the sham TENS treatment and active TENS treatment groups that received the same dose of anesthesia. Each animal received only one TENS treatment, that is, sham TENS, high-frequency TENS, or low-frequency TENS. No animal was treated on multiple days to eliminate potential cumulative effects of TENS.

Experimental Protocol

Baseline compression withdrawal thresholds were measured bilaterally prior to the induction of knee joint inflammation. Animals were deeply anesthetized with 4% halothane for approximately 5 minutes for injection of the knee joint with kaolin and carrageenan. Then they were allowed to wake up and return to their home cages until testing. Compression withdrawal thresholds were reassessed with separate groups of animals at 4 hours, 24 hours, and 2 weeks after the induction of inflammation. At each time period, rats were randomly assigned to receive sham TENS, high-frequency TENS, or low-frequency TENS. Animals were lightly anesthetized with 1% to 2% halothane for placement of the electrodes and the 20-minute application of TENS, for a total duration of approximately 25 minutes. Compression withdrawal thresholds were assessed after recovery from anesthesia, approximately 15 minutes after the removal of TENS.

The animals were divided into the following groups: (1) 4 hours after the induction of inflammation: placebo, n=8; low-frequency TENS, n=4; and high-frequency TENS, n=4; (2) 24 hours after the induction of inflammation: placebo, n=7; low-frequency TENS, n=8; and high-frequency TENS, n=9; and (3) 2 weeks after the induction of inflammation: placebo, n=8; low-frequency TENS, n=4; and high-frequency TENS, n=4.

Data Analysis

Data were analyzed with a multivariate analysis of variance followed by post hoc testing for differences across time within groups with a paired t test and between groups with an independent t test. Data were converted to percent inhibition with the following formula: [(post-TENS--pre-TENS)/(baseline--pre-TENS)] x 100. Full reversal of hyperalgesia was 100%, that is, equal to baseline values. No change in hyperalgesia was 0%, that is, equal to pre-TENS (inflammation) values. Anything above 100% was analgesic, that is, greater than baseline values. Data are expressed as the mean [+ or -] standard error of the mean (SEM).

Results

Injection of kaolin and carrageenan into the knee joint significantly decreased the compression withdrawal threshold of the knee joint at 4 hours, 24 hours, and 2 weeks after the induction of inflammation (P<.05, paired t test) (Fig. 1). The decreases in the compression withdrawal threshold observed at 4 hours and at 24 hours after the induction of inflammation were significantly greater than that observed at 2 weeks after the induction of inflammation (F=30; df=2,52; P=.0001); the decreases at 4 hours and at 24 hours after the induction of inflammation were similar.

[FIGURE 1 OMITTED]

In the group of animals treated with TENS at 4 hours after the induction of inflammation, there was a significant effect for changes in the compression withdrawal threshold with time (F=118.7; df=2,26; P=.0001). There were decreases in the compression withdrawal threshold from baseline both at 4 hours and after treatment in all groups (P<.05, paired t test). However, treatment with either high-frequency TENS or low-frequency TENS had no effect on the decreased compression withdrawal threshold of the knee joint induced by inflammation compared with the results obtained with sham treatment or before TENS treatment (Fig. 2).

[FIGURE 2 OMITTED]

In the group of animals treated with TENS at 24 hours after inflammation, there was a significant effect for changes in the compression withdrawal threshold with time (F=93.2; df=2,42; P=.001). There were significant decreases from baseline at 24 hours after treatment (Fig. 2). There was a significant effect for changes in the compression withdrawal threshold after treatment with TENS ([F.sub.2,42]=3.8, P=.01). Treatment with high-frequency TENS or low-frequency TENS significantly increased the compression withdrawal threshold compared with the results obtained before TENS treatment (P<.05, paired t test) or with sham treatment (P<.05, Duncan test).

In the group of animals treated with TENS at 2 weeks after inflammation, there was a significant effect for changes in the compression withdrawal threshold with time (F=45.2; df=2,22; P=.001). There were significant decreases from baseline at 2 weeks after treatment (Fig. 2). There was a significant effect for treatment with TENS (F=6.8; df= 2,22; P=.001). Treatment with low-frequency TENS resulted in significantly greater compression withdrawal thresholds than did sham treatment (P<.05, Duncan test). A significant increase in compression withdrawal thresholds occurred after treatment with both high-frequency TENS and low-frequency TENS compared with the results obtained before TENS treatment (P<.05, paired t test).

Figure 3 shows the effects of TENS on the decreased compression withdrawal thresholds of the knee joint (as a measure of hyperalgesia) as percent inhibition (100%=full reversal of hyperalgesia). There were significant effects for changes in the percentage of inhibition of hyperalgesia by TENS (F=8.3; df=2,52; P=.001), for changes in the percentage of inhibition after the induction of inflammation (F=26.1; df=2,53; P=.001), and for an interaction between treatment with TENS and time after the induction of inflammation (F=4.0; df=4,53; P=.007). There was a significant inhibition of hyperalgesia at 24 hours and 2 weeks after treatment with both low-frequency TENS and high-frequency TENS compared with the results obtained with sham treatment (P<.05, Duncan test).

[FIGURE 3 OMITTED]

Discussion

The data from the present study show that injection of kaolin and carrageenan into the knee joint produces a decrease in the compression withdrawal threshold of the inflamed knee joint for up to 2 weeks after induction. This decrease in the compression withdrawal threshold is interpreted as primary hyperalgesia and is greatest at 4 and 24 hours after the induction of inflammation. The data also show that TENS at both high and low frequencies completely reverses the primary hyperalgesia of the inflamed knee joint at 24 hours and 2 weeks after the induction of inflammation but not at 4 hours after the induction of inflammation. These data suggest that TENS inhibits the primary hyperalgesia associated with inflammation in a time--dent manner after inflammation has already developed during both acute and chronic stages. Maximal inflammation occurs at 24 hours after injection of the knee joint with kaolin and carrageenan. (5) However, maximal hyperalgesia occurs within 4 hours after the induction of inflammation. Joint nociceptors and dorsal horn neurons show increased responsiveness within hours after induction that continues to increase as the inflammation develops. (7-10)

The lack of an effect of TENS on primary hyperalgesia at 4 hours, but the reversal of primary hyperalgesia at 24 hours and 2 weeks, was surprising. It is likely that different mechanisms mediate the neuronal responses and the consequent hyperalgesia observed within the first few hours after the induction of inflammation and those that occur at 24 hours or 2 weeks later. In the peripheral nervous system, time-dependent changes are observed in dorsal root ganglion neurons, with early increases in the number of sodium channels TRPV1. (17) followed by increases in the number of vannilloid channel TRPV1.17 The levels of glutamate are increased in the dorsal horn of the spinal cord for 24 hours after the induction of knee joint inflammation, the levels of substance P remain increased for at least 1 week after the induction of inflammation, (18) and the levels of the transcription factor c-fos initially increase in the acute stages of inflammation and then decrease in the chronic stages. (19)

There are time-dependent changes in the activation of supraspinal pathways that modulate nociception after inflammation such that within hours, there is strong facilitation of inflammatory hyperalgesia; this response is followed 24 hours later by increased inhibition of hyperalgesia that decreases in the chronic stages. (20,21) A strong facilitatory influence early in the hyperalgesic phase could override the activation of inhibitory pathways by TENS and could result in TENS being ineffective for primary hyperalgesia. In the later phases, TENS might potentiate and prolong the increased inhibition observed after inflammation and therefore result in a reduction in primary hyperalgesia. Thus, the time-dependent dent effect of TENS on primary hyperalgesia likely depends on the time-dependent neuronal changes in both the peripheral and the central nervous systems.

These data extend the results of a previous study that examined the effects of TENS on the primary hyperalgesia observed at 4 hours after carrageenan-induced paw inflammation. (14) In that study, high-frequency TENS partially reversed the hyperalgesia, whereas low-frequency TENS was ineffective. (14) With the same model, however, treatment of the paw at 2 hours after the induction of inflammation and before the development of hyperalgesia prevented the onset of primary mechanical hyperalgesia of the paw. (22) The carrageenan-induced paw inflammation model is a model of cutaneous inflammation with sensitization of cutaneous nociceptors. (23)

In contrast, the model used in the present study does not result in cutaneous inflammation, (18) and joint nociceptors are sensitized after the occurrence of inflammation. (24) Differences between cutaneous inflammation and joint inflammation, between the timing of TENS treatments (2 hours, 4 hours, 24 hours, or 2 weeks), or between the tissues tested (cutaneous or joint) could account for the differences in effectiveness.

Previously it was shown that both low-frequency TENS and high-frequency TENS are equally effective in reducing secondary hyperalgesia at 4 hours and 24 hours after joint inflammation (11,12,25) and at 2 weeks after muscle inflammation. (13) The mechanisms of the reduction of hyperalgesia by TENS involve peripheral, spinal, and supraspinal sites. (26-30) Different neurotransmitters and receptors mediate the effects of high-frequency TENS and low-frequency TENS. Low-frequency TENS releases serotonin spinally and activates the serotonin receptors, 5-HT2 and 5-HT3, muscarinic and mu-opioid receptors in the spinal cord, and mu-opioid receptors supraspinally. (26-29,31,32) High-frequency TENS, on the other hand, releases gamma-aminobutyric acid (GABA) spinally, decreases glutamate levels spinally, and activates delta-opioid and muscarinic receptors in the spinal cord and delta-opioid receptors supraspinally. (26-29,31,33) Thus, TENS reduces hyperalgesia through endogenous neurotransmitters and their receptors primarily at sites in the central nervous system. It is expected that TENS will reduce the sensitization of dorsal horn neurons (34,35) through the activation of these receptors to result in a reduction in hyperalgesia both at the site of injury and outside the site of injury.

Previous studies from our laboratory and others showed that neither high-frequency TENS nor low-frequency TENS affects the swelling that results from carrageenan-induced paw inflammation or kaolin- and carrageenan-induced knee joint inflammation. (11,22) In contrast, other investigators showed that pulsed monophasic cathodal stimulation (high-voltage pulsed current [HVPC]) with a pulse duration of 13 microseconds (twin peaks of 5 and 8 microseconds) limits edema in frogs and rats with either crush injury or hyperflexion injury. (36-40) The differences among the above-described experiments may be attributable to differences in parameters of stimulation, species, electrode application, or timing of application. Transcutaneous electrical nerve stimulation uses a symmetrical or asymmetrical biphasic waveform, which does not allow for a buildup of charge under the electrodes. In contrast, HVPC uses a monophasic waveform, which results in a buildup of charge under the electrodes. Indeed, prior studies documented consistently that the cathode is required as the treatment electrode to limit edema. (36-40)

The electrical stimulation in the HVPC experiments was applied with the limb immersed in water, (36-40) whereas for TENS-treated animals, small adhesive electrodes were applied to the skin around the site of inflammation. (11) With the immersion technique, the entire limb in contact with the water becomes the treatment electrode, and there would be a slight compressive force as well. Furthermore, HVPC was found to be ineffective in reducing edema in Sprague-Dawley rats but effective in edema reduction in Zucker-Lean and Brown Norway rats. (40) Our earlier TENS studies (11-14,25-29,31-34) were done with Sprague-Dawley rats. Therefore, there may be a species-related effect of electrical stimulation on edema. Finally, the timing of the application in the TENS studies was distinctly different from that in the HVPC studies.

In the HVPC experiments in which a reduction in edema was noted, the electrical stimulation was applied in the first 10 minutes following crush injury or hyperflexion injury of the animal limb. (36-40) The treatment duration was usually 4 bouts of 30 minutes of stimulation followed by 30 minutes of rest for up to 4 hours. In the TENS studies, the electrical stimulation was applied after the development of inflammation at 4 hours, 24 hours, or 2 weeks, and only 1 treatment was given. In an earlier study, Mohr et al (41) did not find a significant difference between control and Sprague-Dawley rats treated with HVPC when electrical stimulation was applied at 24 hours after inflammation was induced by impact injury. In addition, these animals did not receive the water immersion treatment protocol. (41) Thus, HVPC may be a better treatment than TENS for the reduction of inflammation, particularly if given early during the development of inflammation. In contrast, TENS provides effective relief of pain and hyperalgesia. It is clear that the decrease in hyperalgesia observed in our experiments is unrelated to a decrease in edema but is related to a modulation of nociceptive information.

Clinically, TENS is used to reduce pain at the site of injury. Systematic reviews have shown that both low-frequency TENS and high-frequency TENS reduce pain and improve function in people with knee joint osteoarthritis) Transcutaneous electrical nerve stimulation increases gait speed and gait distance and decreases pain dtLring walking in people with postsurgery abdominal pain. (42) Furthermore, TENS reduces analgesic intake, decreases side effects of opioids, and improves quality of life. (43-46) However, previous studies did not investigate the effects of TENS on primary or secondary hyperalgesia in human subjects. We propose that TENS may be more effective for pain with movement and decrease analgesic intake, properties that would result in improved function and quality of life. Future clinical studies should confirm these conclusions by measuring effects on multiple outcomes, including primary hyperalgesia and secondary hyperalgesia, and at different time periods, including times of acute inflammation and chronic inflammation.

Conclusion

In summary, the present preclinical study showed that TENS was ineffective in reducing primary hyperalgesia in the early, acute phase of inflammation, 4 hours after induction. However, of note, secondary hyperalgesia was reduced in this early, acute phase of inflammation. (11) Thus, clinically, TENS could be effective in reducing radiating pain and secondary hyperalgesia but likely not primary hyperalgesia shortly after injury. However, 24 hours later, when inflammation was still acute, and 2 weeks later, when inflammation was chronic, TENS reduced both primary hyperalgesia and secondary hyperalgesia. Thus, clinically, after the early, acute phase of inflammation, TENS may be more effective in reducing pain and hyperalgesia.

Ms Vance, Dr Radhakrishnan, and Dr Sluka provided concept/idea/research design. All authors provided data collection. Dr Sluka provided writing, fund procurement, and facilities/equipment. Ms Vance and Dr Sluka provided data analysis, project management, and clerical support. Ms Vance, Dr Radhakrishnan, and Dr Skyba provided consultation (including review of manuscript before submission). The authors thank Ms Amy Russell and Ms Tammy Lisi for their technical assistance and Ms Carol Leigh for excellent secretarial assistance.

All experiments were approved by the Animal Care and Use Committee at the University of Iowa and were conducted in accordance with the National Institutes of Health guidelines for the ethical treatment of animals.

The study was funded by National Institutes of Health grant K0202201.

This research, in part, was presented as a poster at the Combined Sections Meeting of the American Physical Therapy Association; February 1-5, 2006; San Diego, Calif. An oral presentation of the findings was made at the World Congress on Pain; August 21-26, 2005; Sydney, New South Wales, Australia.

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* Empi Inc, 599 Cardigan Rd, St Paul, MN 55126-4099.

CGT Vance, PT, MA, is Associate, Graduate Program in Physical Therapy and Rehabilitation Science, University of Iowa, Iowa City, Iowa.

R Radhakrishnan, PhD, was Research Investigator, Pain Research Program and Graduate Program in Physical Therapy and Rehabilitation Science, University of Iowa, at the time of the study. He is currently affiliated with the College of Pharmacy, Western University of Health Sciences, Pomona, Calif.

DA Skyba, DC, PhD, is Assistant Professor of Neuroscience, Department of Basic Sciences, Palmer College-Florida, Port Orange, Fla.

KA Sluka, PT, PhD, is Professor, Pain Research Program and Graduate Program in Physical Therapy and Rehabilitation Science, 1-252 MEB, University of Iowa, Iowa City, IA 52242 (USA). Address all correspondence to Dr Sluka at: [e-mail].

[Vance CGT, Radhakrishnan R, Skyba DA, Sluka KA. Transcutaneous electrical nerve stimulation at both high and low frequencies reduces primary hyperalgesia in rats with joint inflammation in a time-dependent manner. Phys Ther. 2007;87:44-51.]

[C] 2007 American Physical Therapy Association

This article was received January 30, 2006 and was accepted August 11, 2006.