High-frequency, but not low-frequency ... - Wiley Online Library

Journal of Neurochemistry, 2005, 95, 1794–1801

doi:10.1111/j.1471-4159.2005.03511.x

High-frequency, but not low-frequency, transcutaneous electrical nerve stimulation reduces aspartate and glutamate release in the spinal cord dorsal horn K. A. Sluka, C. G. T. Vance and T. L. Lisi Physical Therapy and Rehabilitation Science Graduate Program, University of Iowa, Iowa City, Iowa, USA

Abstract Transcutaneous electrical nerve stimulation (TENS) is a commonly utilized non-pharmacological treatment for pain. Studies show that low- and high-frequency TENS utilize opioid, serotonin and/or muscarinic receptors in the spinal cord to reduce hyperalgesia induced by joint inflammation in rats. As there is an increase in glutamate and aspartate levels in the spinal cord after joint inflammation, and opioids reduce glutamate and aspartate release, we hypothesized that TENS reduces release of glutamate and aspartate in animals with joint inflammation by activation of opioid receptors. Using microdialysis and HPLC with fluorescence detection, we examined the release pattern of glutamate and aspartate in the dorsal horn in response to either low-frequency (4 Hz) or high-frequency (100 Hz) TENS. We

examined the effects of TENS on glutamate and aspartate release in animals with and without joint inflammation. Highfrequency, but not low-frequency, TENS significantly reduced spinal glutamate and aspartate in animals with joint inflammation compared with levels in those without joint inflammation. The reduced release of glutamate and aspartate by highfrequency TENS was prevented by spinal blockade of deltaopioid receptors with naltrindole. Thus, we conclude that high-frequency TENS activates delta-opioid receptors consequently reducing the increased release of glutamate and aspartate in the spinal cord. Keywords: excitatory amino acid, hyperalgesia, microdialysis, opioid, pain, spinal cord. J. Neurochem. (2005) 95, 1794–1801.

Transcutaneous electrical nerve stimulation (TENS) is a commonly utilized non-pharmacological treatment for pain. Even though the literature on clinical application of TENS is extensive, surprisingly few reports have addressed the neurobiological basis for the effectiveness of TENS. TENS was initially reported for pain relief in 1967 by Wall and Sweet as a clinical test for implantation of dorsal column stimulators and was based on mechanisms described in the gate control theory of pain (Melzack and Wall 1965). The gate control theory proposes that pain reduction occurs at the segmental level because stimulation of large-diameter afferent fibers (Aa and Ab fibers) activates local inhibitory circuits in the dorsal horn of the spinal cord, and thus alters responses evoked by nociceptive fibers (C and Ad). In support of this, TENS reduces the nociceptive responses and inflammationinduced sensitization of dorsal horn neurons (Lee et al. 1985; Garrison and Foreman 1994, 1997; Ma and Sluka 2001). Furthermore, the effects of high-frequency TENS remain after spinal transection (Woolf et al. 1977, 1980) pointing to a segmental site of action within the spinal cord. However, the effects of TENS in rats with spinal transection are reduced

compared with those in intact rats (Woolf et al. 1977, 1980) suggesting that the anti-hyperalgesic effects of TENS may additionally involve bulbospinal pathways. More recent data show that TENS activates opioid receptors spinally and supraspinally to reduce hyperalgesia associated with joint inflammation (Sluka et al. 1999; Kalra et al. 2001). In the spinal cord, a complicated neurochemistry involves activation of acetylcholinergic and serotonergic receptors by TENS to reduce hyperalgesia (Radhakrishnan et al. 2003a; Radhakrishnan and Sluka 2003). Joint inflammation results in increased extracellular concentrations of the excitatory neurotransmitters glutamate and aspartate in the spinal cord dorsal horn (Sluka and Westlund

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Received July 8, 2005; revised manuscript received August 2, 2005; accepted August 29, 2005. Address correspondence and reprint requests to Kathleen Sluka, Physical Therapy and Rehabilitation Science Graduate Program, University of Iowa, Iowa City, IA 52242, USA. E-mail: [email protected] Abbreviations used: ACSF, artificial cerebrospinal fluid; TENS, transcutaneous electrical nerve stimulation.

Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 95, 1794–1801

TENS reduces glutamate release 1795

1992; Sorkin et al. 1992). Previous studies showed that spinal application of opioid agonists reduces glutamate concentrations in the spinal cord and hyperalgesia associated with inflammation (Malmberg and Yaksh 1995; Ueda et al. 1995; Yu et al. 2002; Skyba et al. 2005). Furthermore, peripheral inflammation up-regulates mu- and delta-opioid receptors in the spinal cord and opioid agonists are more effective (Cahill et al. 2003a, 2003b; Stanfa and Dickenson 1994). Because TENS inhibits inflammation-induced hyperalgesia by activation of opioid receptors, we hypothesized that TENS produces its anti-hyperalgesic effect by reducing extracellular concentrations of glutamate and aspartate in the spinal cord through activation of opioid receptors. We further hypothesized that this reduction in extracellular glutamate concentrations would be specific for the animal with joint inflammation and would not occur an uninjured animal as there is an increased release of glutamate after joint inflammation. Materials and methods All experiments were approved by the Animal Care and Use Committee at the University of Iowa. Injection of kaolin and carrageenan into the knee joint All experiments were performed in 250–350 g Sprague–Dawley rats (Harlan, Indianapolis, IN, USA) with knee joint inflammation. A mixture of 3% kaolin and 3% carrageenan (0.1 mL in sterile saline, pH 7.2–7.4) was injected into both knees while the rat was anesthetized with halothane (2–5%) (Sluka and Westlund 1993) at the time the microdialysis fiber was placed, the day before collection of samples for analysis. Application of TENS Electrodes were placed on the medial and lateral aspects of both knee joints before collecting samples. Either high-frequency (100 Hz) or low-frequency (4 Hz) stimulation was used. All other parameters – pulse width (100 ls), amplitude/intensity, sensory level (just below motor contraction), duration (20 min) – were kept constant. These parameters were the same as those shown previously to reduce heat and mechanical hyperalgesia in this animal model of joint inflammation (Sluka et al. 1998; King and Sluka 2001), and are similar to those utilized clinically. The TENS units used in these studies are clinically available and were donated by EMPI (EMPI Eclipse+, Minneapolis, MN, USA). The waveform is a balanced asymmetrical biphasic square wave. Amplitude is adjustable from 0 to 60 mA, pulse width is adjustable from 30 to 250 lsec, and pulse rate (frequency) is adjustable from 2 to 125 Hz. Electrodes are 12.7 mm diameter round pregelled and used clinically for TENS treatment of small areas such as the hand or fingers. The size of electrodes used in the proposed experiments was equivalent to the area of tissue that would be covered by electrodes in human subjects receiving TENS to the knee joint. Placement of microdialysis probes in the spinal cord Microdialysis fibers (200 lm external diameter; Hospal Filtral AN69, Lakewood, CO, USA) were covered with epoxy, except for a

2-mm gap, and placed the day before the experiment in male Sprague–Dawley rats as described previously (Sluka and Westlund 1992). Rats were anesthetized with 2–5% halothane during microdialysis fiber placement. The T13 vertebra was cleared of muscle and small holes were drilled into the lateral aspect on each side to expose a small portion of the spinal cord. Microdialysis fibers were inserted transversely across the dorsal horn of the spinal cord through the two holes and then fixed to the bone with dental cement. The free ends of the microdialysis fiber were then inserted into PE20 tubing and the connection secured with epoxy. The incision was sutured closed and animals were allowed to recover for 24 h. On the day of the experiment, animals were anesthetized initially with 50 mg/kg i.p. sodium pentobarbital and maintained with 2–4 mg/kg/h i.v. sodium pentobarbital. The depth of anesthesia was maintained constant by examining eye blink every 10 min at the time of sample collection. Artificial cerebrospinal fluid (ACSF) was infused at 5 lL/min throughout the experiment. All samples were collected on ice, immediately frozen on dry ice, and stored at ) 70°C until analysis. Methylene blue was infused through the microprobe for 10 min after the collection of the last sample. At the end of the experiment, rats were killed with an overdose of sodium pentobarbital, and spinal cords were removed, fixed in 10% formalin and cut on a cryostat at 50 lm for analysis of probe placement. Application of naltrindole To test whether the decreases in glutamate and aspartate that occur after high-frequency TENS depend on activation of delta-opioid receptors, we perfused naltrindole (1 mM) through the microdialysis fiber for 1 h before and during application of TENS. Two 10-min baseline samples were collected with ACSF before starting the naltrindole perfusion, and four 10-min samples were collected during naltrindole perfusion and before the application of TENS. Two 10-min samples were then collected during the 20-min TENS application while naltrindole was perfused through the microdialysis fiber. After treatment with TENS, ACSF was perfused for 40 min, and four 10-min samples collected. The dose and delivery method for naltrindole were identical to those in previous experiments in which we showed a blockade of the anti-hyperalgesic effects of high-frequency TENS in behavioral studies (Sluka et al. 1999). We previously showed that this dose of naltrindole in the spinal cord blocks delta-opioid, but not mu- or kappa-opioid, receptors (Sluka et al. 1999). HPLC Samples from microdialysis experiments were analyzed for glutamate using fluorescence detection with precolumn o-phthaldialdehyde (Sigma, St Louis, MO, USA) derivitization (Zahn et al. 2002). All samples were stored at ) 70°C until analysis. A 20-lL aliquot of sample was diluted in 180 lL deionized water containing 1 ng/mL of the internal standard homoserine. A refrigerated autoinjector set at 8°C was used for precolumn derivitization with 50 lL o-phthaldialdehyde. Immediately following derivitization 200 lL of each sample was injected on to the column. The column was a supelcosil LC-18 HPLC column (5 lm particle diameter, 4.6 mm internal diameter, 15 cm long) with corresponding guard column. The mobile phase consisted of 17% methanol and 0.05 M sodium acetate flowing at approximately 1 mL/min; flow rates were optimized for each run to obtain the best resolution of analytes.


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The fluorescence detector was set at 330 nm for excitation and 420 nm for emission. Standards were dispersed through the run so that there were three standards at the beginning of the run, one standard every six samples, and two standards at the end of the run. The limit of detection for glutamate was 0.08 ng/lL, the limit of quantification was 0.10 pg/mL and precision was 5%. Concentrations were calculated based on a standard curve. Statistical analysis Data were analyzed with a repeated measures ANOVA for differences between groups (inflamed vs. non-inflamed) and frequency (0, 4 and 100 Hz), followed by Tukey’s post hoc test. Data were converted to a percentage of baseline for analysis of differences between groups; baseline was 100%. Difference in summary responses during and after TENS were analyzed with a one-way ANOVA for inflamed and non-inflamed sets, followed by Tukey’s post hoc test. Differences before and after naltrindole perfusion were tested with a paired t-test. p < 0.05 was considered significant. Data are represented as the mean ± S.E.M.

Results

Baseline concentrations of aspartate in the dorsal horn of the spinal cord were similar in the groups with joint inflammation and those without. The baseline concentrations averaged 1.8 ± 0.39 ng/mL in animals without joint inflammation and 2.9 ± 0.56 ng/mL in animals with inflammation (p ¼ 0.12). A trend for an increase in glutamate concentrations in the dorsal horn occurred 24 h after inflammation (p ¼ 0.06); concentrations in the noninflamed group and the group with joint inflammation were 14.2 ± 4.2 and 28.4 ± 6.1 ng/mL respectively. Application of high-frequency TENS reduced glutamate and aspartate concentrations in the dorsal horn of the spinal cord when microdialysis fibers were placed in the L3 or L4 spinal segment of rats in animals with knee joint inflammation. There was a significant effect of time for glutamate (F1,38 ¼ 4.1, p ¼ 0.05) and aspartate (F1,38 ¼ 5.3, p ¼ 0.03). A significant difference in glutamate (F1,9 ¼ 6.9, p ¼ 0.03) and aspartate (F1,9 ¼ 9.6, p ¼ 0.01) concentrations was noted in animals with joint inflammation treated with high-frequency TENS compared with those that did not receive TENS. There was also a significant difference in glutamate (F1,11 ¼ 4.2, p ¼ 0.05) and aspartate (F1,11 ¼ 5.1, p ¼ 0.04) concentrations between animals with and without inflammation subjected to high-frequency TENS. In animals without joint inflammation TENS had no effect on glutamate and asparate concentrations. Thus, TENS was only effective in animals with joint inflammation. In the animals with joint inflammation, analysis of the summary responses revealed a significant effect during treatment with TENS but not after treatment with TENS for both glutamate (F2,26 ¼ 4.7, p ¼ 0.02) and aspartate (F2,26 ¼ 3.5, p ¼ 0.04) in the dorsal horn. Significant decreases were noted in the group that received high-

frequency TENS compared with those that did not receive TENS (glutamate, p ¼ 0.02; aspartate, p ¼ 0.04). However, after application of TENS there was no significant difference between animals with or without joint inflammation. In some animals, microdialysis fibers were inadvertently placed in the L5 or L6 spinal segments. No change in glutamate or aspartate occurred when microdialysis fibers were placed in the L5 or L6 segment of the spinal cord in animals with joint inflammation (n ¼ 2) or those without inflammation (n ¼ 3) (data not shown). In contrast, application of low-frequency TENS had no effect on glutamate and aspartate concentrations in the dorsal horn of the spinal cord in animals with (n ¼ 11) or without (n ¼ 5) joint inflammation (Figs 1 and 2) in animals with microdialysis fibers placed in the L3 or L4 spinal segment of rats. Glutamate and aspartate concentrations remained at baseline levels throughout the sampling period in the group that received sham TENS in both the inflamed (n ¼ 7) and non-inflamed (n ¼ 7) groups. Treatment of the spinal cord for 1 h with naltrindole to block delta-opioid receptors significantly increased glutamate (p ¼ 0.03) and aspartate (p ¼ 0.02) concentrations compared with baseline levels in animals with knee joint inflammation (Fig. 3). Naltrindole prevented the decrease in glutamate (p ¼ 0.01) and aspartate (p ¼ 0.05) concentrations that normally occur during TENS. Figure 3 shows the percentage increase in glutamate and aspartate concentrations in the dorsal horn during naltrindole perfusion and during naltrindole perfusion in combination with TENS in the same animals. For comparison, the bottom panel shows the percentage of baseline in animals with inflammation treated with high-frequency TENS (Fig. 3). Discussion

High-frequency TENS significantly decreased aspartate and glutamate concentrations in the extracellular fluid of the dorsal horn of the spinal cord in animals with joint inflammation but not in those without inflammation. This decrease was prevented by application of naltrindole before application of TENS, suggesting that TENS decreases glutamate and aspartate concentrations in rats with inflammation through activation of delta-opioid receptors. Lowfrequency TENS had no effect on the aspartate and glutamate concentrations in the dorsal horn of the spinal cord. The present study shows that the reduction of aspartate and glutamate concentrations by high-frequency TENS only occurs after joint inflammation, and not in rats without inflammation. These data further show that different mechanisms account for electrical stimulation analgesia in animals with inflammation compared with normal animals. After joint inflammation glutamate and aspartate concentrations in the spinal dorsal horn are increased (Sluka and Westlund 1992), probably resulting in sensitization of dorsal horn



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Fig. 1 Time course of glutamate responses in groups of rats with or without inflammation that did not receive TENS, received low-frequency TENS or received high-frequency TENS (left panels). The right-hand panels summarize the percentage change from baseline

during and after TENS. Data are mean ± SEM. *p < 0.05 versus no TENS inflamed group and high-frequency TENS non-inflamed group (Tukey’s test).

neurons (Dougherty et al. 1992) and hyperalgesia (Sluka and Westlund 1993; Radhakrishnan et al. 2003b). Indeed deltaopioid agonists show increased potency after inflammation (Hylden et al. 1991; Stanfa et al. 1992), and there is increased delta-opioid receptor expression on the cell membranes of dorsal horn neurons (Cahill et al. 2003b). The majority (70%) of delta-opioid receptors in the spinal cord are eliminated by dorsal rhizotomy (Abbadie et al. 2002) suggesting that delta-opioid receptors are located presynaptically; however, 30% remain after dorsal rhizotomy. Thus, an electrical stimulation technique that activates endogenous delta-opioid receptors would be expected to show increased efficacy for pain relief after inflammation. We hypothesize that high-frequency TENS reduces initial release of glutamate and aspartate from sensitized primary afferent fibers, and prevents subsequent release from interneurons in the dorsal horn. High-frequency TENS reduces hyperalgesia and dorsal horn neuron sensitization induced by carrageenan inflammation (Sluka et al. 1998; Gopalkrishnan and Sluka 2000; King

and Sluka 2001; Ma and Sluka 2001). Specifically, secondary and primary hyperalgesia to heat and mechanical stimuli induced by joint inflammation is significantly reduced by one 20-min application of high-frequency TENS. The sensitization of high-threshold and wide dynamic range dorsal horn neurons to noxious and/or innocuous stimuli is also completely reduced by high-frequency TENS. Furthermore, the pharmacology of this inhibition has been investigated and includes peripheral, spinal and suprapinal sites (Sluka et al. 1999; Kalra et al. 2001; Radhakrishnan and Sluka 2003; Radhakrishnan et al. 2003a; King et al. 2005). Previous studies on the spinal cord showed that blockade of deltaopioid and M1 and M3 muscarinic receptors in the spinal cord prevents the anti-hyperalgesic effects of high-frequency TENS in this model of joint inflammation (Sluka et al. 1999; Radhakrishnan and Sluka 2003). This is consistent with earlier work showing that activation of delta-opioid receptors in the spinal cord reduces evoked release of glutamate and aspartate (Ueda et al. 1995), dorsal horn neuron sensitization (Stanfa et al. 1992) and hyperalgesia (Stewart and Hammond



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Fig. 2 Graphs show the time course of aspartate responses in groups of rats with or without inflammation that did not receive TENS, received low-frequency TENS or received high-frequency TENS (left panels). The right-hand panels summarize the percentage change

from baseline during and after TENS. Data are mean ± SEM. *p < 0.05 versus no TENS inflamed group and high-frequency TENS non-inflamed group (Tukey’s test).

1994) associated with inflammation. Similarly, dorsal column stimulation at high frequencies reduces glutamate and aspartate concentrations in neuropathic rats with allodynia (Cui et al. 1997). Thus, we propose that activation of deltaopioid receptors during TENS reduces excitatory amino acid release, and the consequent dorsal horn neuron sensitization and hyperalgesia. The lack of effect of low-frequency TENS on glutamate and aspartate was surprising, and suggests that low- and highfrequency TENS involve different mechanisms. Indeed previous data showed that low-frequency TENS utilizes serotonin 5-HT2 and 5-HT3 and mu-opioid receptors in the spinal cord to reduce hyperalgesia induced by joint inflammation (Sluka et al. 1999; Radhakrishnan et al. 2003a). Blockade of delta-opioid receptors in the spinal cord has no effect on low-frequency TENS anti-hyperalgesia (Sluka et al. 1999). Similarly, in animals without joint inflammation, analgesia produced by low-frequency TENS is prevented by non-selective blockade of serotonin receptors with methys-

ergide (Shimizu et al. 1981), and there is increased release of met-enkephalin-arg-phe in the CSF (Han et al. 1991). Mu-opioid receptors are located presynaptically on primary afferent fibers and postsynaptically on laminae II glutamatergic neurons (Arvidsson et al. 1995; Trafton et al. 2000; Abbadie et al. 2002). Pharmacological studies have shown that evoked release of glutamate in spinal cord slices is reduced by mu-opioid agonists (Ueda et al. 1995; Kangrga and Randic 1991). We propose that TENS activates endogenous circuitry to produce release of neurotransmitters resulting in an analgesic effect that is distinctly different from exogenous activation with agonists applied directly to the spinal cord. Surprisingly, spinal blockade of delta-opioid receptors in animals with joint inflammation increased the concentrations of glutamate and aspartate in the dorsal horn. The concentration and method of delivery were the same as those used in a previous study that showed no effect of naltrindole on behavioral responses in animals with joint inflammation that did not receive TENS (Sluka et al. 1999). Furthermore, this



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Fig. 3 Changes in glutamate and aspartate in the dorsal horn in rats with joint inflammation. The top panel shows the percent of baseline responses during infusion of naltrindole (closed bars) and naltrindole plus TENS (open bars). The bottom panel shows the percent of baseline in animals infused with ACSF (closed bars) compared to the percent of baseline during naltrindole infusion (open bars). A significant difference in percent baseline between animals treated with ACSF and those treated with naltrindole (p < 0.05, t-test) occurred. Data are mean ± S.E.M.

dose of naltrindole was shown to block analgesia produced by a delta-opioid agonist, but not a mu-opioid or kappa-opioid agonist (Sluka et al. 1999). Thus, the present data suggest that there is a tonic inhibition of aspartate and glutamate release by endogenous delta-opioid agonists. They also show that TENS has no effect on the release of aspartate and glutamate during infusion of naltrindole. However, one limitation of these results is that, because aspartate and glutamate concentrations were already increased by naltrin-

dole, it might not have been possible to detect a small decrease in aspartate and glutamate during TENS. Placement of microdialysis fibers into the spinal dorsal horn by itself produces some damage to the dorsal horn. To minimize the acute response to insertion of the microdialysis fibers, we performed all placements 1 day before beginning the experiment. Further control groups were analyzed that did not receive TENS and were infused continuously with ACSF. In these animals, both those with and those without joint inflammation, there was no change in extracellular concentrations of glutamate and aspartate for the entire sampling period. Thus, minimal effects on neurotransmitter release occur as a result of microdialysis fiber insertion. In summary, high-frequency TENS reduces release of excitatory neurotransmitters in the dorsal horn in animals with tissue injury, but not in those without. Low-frequency TENS, on the other hand, has no effect on release of excitatory neurotransmitters in the dorsal horn but rather utilizes inhibitory neurotransmitters associated with supraspinal sites. Understanding the mechanisms of action of TENS will help clinicians in deciding the treatment of choice for people in pain. In several studies conducted in those with osteoarthritis and rheumatoid arthritis, TENS has been shown to be effective with respect to a variety of outcome measures including subjective pain rating scores, loading time to increased pain, analgesic intake, sleep time, knee function tests (range of motion, walking time, tenderness) and morning stiffness. Most studies assessed sensory TENS at either high (50–100Hz) or low (2–4 Hz) frequency. In general, there was a decrease in subjective pain scores with either high- or low-frequency TENS and increased function of the joint after treatment (Mannheimer et al. 1978; Mannheimer and Carlsson 1979; Kumar and Redford 1982; Smith et al. 1983; Fargas-Babjak et al. 1992; Zizic et al. 1995; Cheing et al. 2002, 2003; Law and Cheing 2004), supporting the clinical efficacy of TENS in patients with arthritis. Acknowledgements Supported by a grant from the Arthritis Foundation and National Institutes of Health grant K0202201. TENS units were donated by EMPI, Inc.

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