Anesth Analg 2003;96:1711-1716
© 2003 International Anesthesia Research Society
PAIN MEDICINE
Antinociceptive Potentiation and Attenuation of Tolerance by Intrathecal Electric Stimulation in Rats
Chung-Ren Lin, MD PhD*,
,
Lin-Cheng Yang, MD,
Huey-Ling You, MS*,
Chien-Te Lee, MD
,
Ming-Hong Tai, PhD
,
Ping-Heng Tan, MD,
Ming-Wei Lin, MS, and
Jiin-Tsuey Cheng, PhD*
*Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Departments of
Anesthesiology and
Nephrology, Kaohsiung Chung Gang Memorial Hospital, Kaohsiung, Taiwan; and
Department of Medical Research, Kaohsiung Veteran General Hospital, Kaohsiung, Taiwan
Address correspondence and reprint requests to Jiin-Tsuey Cheng, PhD, Department of Biological Sciences, National Sun Yat-Sen University, 70 Lien-Hai Rd., Kaohsiung, Taiwan 804. Address e-mail to tusya{at}mail.nsysu.edu.tw
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Abstract
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We tested whether intrathecal electric stimulation would reduce the tolerance to chronic morphine use and the severity of precipitated morphine withdrawal. Rats received intrathecal electrode catheter implantation and a continuous intrathecal infusion of morphine (2 nmol/h) or saline for 7 days. Intrathecal electric stimulations (0, 20, or 200 V) were performed once daily during the same period. Daily tail-flick and intrathecal morphine challenge tests were performed to assess the effect of intrathecal electric stimulation on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg/kg) was performed to assess morphine dependence, and changes in spinal neurotransmitters were monitored by microdialysis. The antinociceptive effect of intrathecal morphine was increased by 200 V of electric stimulation. The magnitude of tolerance was decreased in the rats receiving the 2 nmol/h infusion with 200 V of intrathecal electric stimulation compared with the control group (morphine 2 nmol/h alone) (AD50, 13.6 vs 124.7 nmol). The severity of naloxone-induced withdrawal was less in the rats receiving 200 V of stimulation. Intrathecal stimulation thus enhances analgesia and attenuates naloxone-induced withdrawal symptoms in rats receiving chronic intrathecal morphine infusion. Increases in spinal glycine release may be the underlying mechanism. This method may merit further investigation in the context of the long-term use of intrathecal opioids for controlling chronic pain.
IMPLICATIONS: Control of chronic pain is a major health problem. We show here that direct electrical stimulation of the spinal cord in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.
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Introduction
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Control of chronic pain is a worldwide challenge for clinicians. Current treatments are often inadequate and may involve invasive procedures with unacceptable side effects. In cancer pain, IV or oral morphine is only partially effective and is accompanied by side effects such as constipation, sedation, and respiratory depression (1). Furthermore, its practical use is often limited by concerns about tolerance. Catheters can be implanted into the subarachnoid space and connected to infusion pumps to localize delivery of opioids and reduce their side effects (1), but the problems of tolerance and withdrawal are still unsolved.
Spinal cord stimulation has been used extensively during the last 30 yr for the management of certain chronic pain states (2,3). It induces the release of dorsal horn 
-aminobutyric acid (GABA), reduces the release of glutamate and aspartate, and also reduces the release of excitatory amino acid by its action on the GABAB receptor, thus alleviating neuropathic pain (2,4,5). However, the method is poorly effective for the attenuation of morphine tolerance.
Brief high-voltage electric stimulation of the brain increases the GABA level, upregulates GABA receptor messenger RNA, and potentiates 5-hydroxy-tryptamine receptor function (6,7). This method was reported to attenuate withdrawal symptoms caused by chronic systemic opioid administration (8). In chronic spinal opioid-infused rats, opioid tolerance develops to antinociceptive effects in the spinal cord (9,10). Brief high-voltage electric stimulation of the spinal cord causes a variety of neurochemical and neurophysiologic effects, some of which may interact with the pathophysiologic mechanisms in morphine tolerance and dependence. Furthermore, cerebrospinal fluid (CSF) is the most conductive intraspinal element (2). Therefore, an electrical field that reaches the CSF has the greatest potential to be conducted to nearby structures. The goal of our study was to investigate the effects on morphine-induced analgesia of intrathecal high-voltage electric stimulation of the spinal cord in the development of antinociceptive tolerance and dependence to morphine in rats.
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Methods
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This protocol was approved by our animal research and use committee. Male Sprague-Dawley rats (NSC, Taiwan), weighing 300–350 g, were used in our study. To reduce the influences of handling on nociceptive responses, all animals were handled and trained for at least 4 to 6 days before intrathecal catheterization and testing. An electrode-microdialysis probe was constructed by passing a platinum wire (0.066 mm) into the intrathecal catheter of a three-lumen microdialysis probe (9 cm long; Marsala Co.) protruding 5 mm and covered by microdialysis membrane (11,12). The intrathecal electrocatheter was joined to PE-10 polyethylene tubing, PE-10 was joined to PE-20, and PE-20 was joined to PE-60. With rats under 2.5% isoflurane anesthesia, the probe was then implanted into the rats’ intrathecal lumbar (L3-4) space through a cisternal incision. The electrode part was contacted with a negative electrode clamp through the sheath. A positive electrode was placed on the base of the tail. Intrathecal electric stimulation was performed with an electroporator (BTX EC830; Genetronics, San Diego, CA). The catheters were connected to osmotic pumps (Model 2001 delivering 1 µL/min; Alza, Palo Alta, CA), filled with the drug or saline, attached to the PE-60 end of the catheter, and implanted subcutaneously. The catheter was cut for the challenge test on Day 8 after the intrathecal infusion.
Rats were randomly assigned to six groups as follows:
- Group 1 (M0): intrathecal infusion with vehicle only as control.
- Group 2 (M0 + V200): infusion with vehicle and daily intrathecal electric stimulation with a pulse (200 V, 50-ms duration, 950-ms interval, 5 iterations) for 7 days.
- Group 3 (M0 + V20): infusion with vehicle and daily intrathecal electric stimulation with a pulse (20 V, 50-ms duration, 950-ms interval, 5 iterations) for 7 days.
- Group 4 (M2): infusion with 2 nmol/h of morphine only.
- Group 5 (M2 + V200): infusion with 2 nmol/h of morphine and daily intrathecal electric stimulation with a pulse (200 V, 50-ms duration, 950-ms interval, 5 iterations) for 7 days.
- Group 6 (M2 + V20): infusion with 2 nmol/h of morphine and daily intrathecal electric stimulation with a pulse (20 V, 50-ms duration, 950-ms interval, 5 iterations) for 7 days.
The tail-flick (TF) test was used to measure responses to noxious somatic stimuli by monitoring latency to withdrawal from a heat source focused on the dorsal surface of the tail approximately 5 cm from the tip. Lack of occurrence of the TF response in 10 s resulted in termination of the stimulus. The 10-s interval was set as the cutoff time to avoid damage to the tail. Measurements were performed daily to assess each animal’s response to noxious somatic stimuli for 7 days during infusion. This study was performed in a double-blinded fashion until all measurements were completed.
On Day 8, a challenge test with morphine was performed to assess the development of tolerance to morphine. In this test, morphine (0.1, 1, 10, or 100 nmol/10 µL) was intrathecally administered after the determination of baseline values, and TF tests were repeated at 5, 10, 15, 20, 30, 60, 90, 120, and 180 min after injection.
To assess dependency in these animals, 36 rats entered into the withdrawal study. On Day 7 at noon, rats from each group were given 2 mg/kg of intraperitoneal naloxone and observed for 1 h in a circular transparent observation chamber for the presence or absence of signs of withdrawal (13): vocalization in response to light touching with a piece of polyethylene tubing, spontaneous vocalization, abnormal posture, ejaculation, "wet dog" head shakes, or attempts to escape. Each group contained six rats. The observer was blinded to group identity.
For microdialysis, animals were anesthetized with 2.5% isoflurane, and the dialysis catheters were perfused with artificial CSF (ACSF) at a rate of 5 µL/min. ACSF contained (mM) 151.1 Na+, 2.6 K+, 0.9 Mg2+, 1.3 Ca2+, 122.7 Cl-, 21.0 HCO3-, 2.5 HPO4-, and 3.5 dextrose. The ACSF was bubbled with 95% oxygen/5% CO2 before each experiment to adjust the final pH to 7.2. All manipulations were preceded by a 30-min washout period followed by two sample collections (10 min each). Ten microliters of dialysate was assayed with on-line high-performance liquid chromatography (HPLC; HP1100/USA) coupled with a fluorescence detector. The detection sensitivity was up to 10-8 M (5–10 pmol in 10-µL tubes).
All data were analyzed by using commercially available computer software (SPSS Version 9.0; SPSS Inc., Chicago, IL). Biochemical results were analyzed by using a nonparametric analysis of variance (ANOVA) (Kruskal-Wallis test followed by the Mann-Whitney U-test). Comparisons between individual treatment groups and time points were performed with the Wilcoxon signed rank test. To compare groups, a post hoc Fisher’s least significant difference test was used. A P value of <0.05 was considered significant. Spinal neurochemical data were expressed as percentage change from baseline ±SD. Where applicable, data from antinociceptive testing, i.e., absolute latencies or calculated maximal possible effect (% MPE = [postdrug value - baseline value]/[cut-off value - baseline value] x 100) were analyzed with one- or two-way ANOVA to detect differences between groups. When differences were found, these findings were subjected to the Scheffé F test (significant at 95%). Unless stated otherwise, single points of comparison were made by using a standard paired or unpaired Student’s t-test. By using linear regression, calculation of the AD50 (95% confidence intervals [CI]) test for relative potency was done where applicable. The tolerance ratio (the ratio of AD50 in treated animals to AD50 of saline-infused animals) and 95% CI were calculated. Differences yielding critical values corresponding to P < 0.05 were considered statistically significant.
For histopathological analysis of the spinal cords, rats were perfusion-fixed with 4% paraformaldehyde. The L2 to L5 spinal cord segments were dissected and postfixed in 1% buffered OsO4 and embedded in Araldite. Ten subserial semithin sections (1 µm thick) were then cut and stained with p-phenylenediamine. Sections were evaluated for the presence of dark-staining neurons, vacuolized degeneration, or both.
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Results
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We prepared 82 animals, and 10 animals were excluded from the study (5 because of implantation failure, 3 because of pump failure, and 2 because of infection). Results from 72 animals (6 animals in each group for microdialysis study and the other 6 in each group for the challenge test) were analyzed.
Baseline values of the % MPEs in the TF test were not statistically different among the groups. In saline-infused groups, there was no significant difference between latencies on Days 1 and 7 in rats that were tested daily (5.7 ± 1.4 s versus 5.1 ± 1.6 s; n = 6; P > 0.1). There was also no significant difference among the three saline-infused groups (M0, M0 + V200, and M0 + V20) (ANOVA; P > 0.1), demonstrating no significant effect after Day 1 from implantation, infusion of the vehicle saline, or intrathecal electric stimulation alone (Fig. 1).
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Figure 1. Time course of the changes in thermal antinociception, percentage of maximal possible effect (% MPE), as measured by the tail-flick test in rats receiving continuous intrathecal infusions. Data are presented as means ± SD (n = 6). Please see the text for details of treatment groups. *Different from morphine 2 nmol/h on that day; P < 0.05. +Different from Day 2 value within the group; P < 0.05.
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Thermal withdrawal responses to morphine at 2 nmol/h on Day 1 (M2; assessed by % MPE) reflected 67% antinociception, but by Days 3–7, antinociception was significantly reduced compared with Day 1 (Fig. 1). Compared with M2, the combination of 200 V of intrathecal electric stimulation and M2 (M2 + V200) resulted in a significant increase in % MPE on Days 1–7, whereas the combination of 20 V of intrathecal electric stimulation with M2 (M2 + V20) did not show a significant difference compared with M2 (Fig. 1).
In the naloxone-induced withdrawal tests, none of the rats infused with saline (n = 6) showed any of the 6 withdrawal signs examined. In contrast, morphine-infused rats (n = 6) all exhibited 3 or more of the 6 signs of withdrawal assessed. The 200-V electroporation and morphine-infused rats (n = 6) showed a significant reduction in 4 of the 6 signs of with-drawal—vocalization to air motion or light touch, spontaneous vocalization, paw lifting, and head shaking—when compared with morphine-infused rats (P < 0.05). The group that received 20 V of intrathecal electric stimulation and morphine-infused rats (n = 6) showed reduction in one sign of withdrawal, i.e., head shaking, when compared with morphine-infused rats (Fig. 2).
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Figure 2. Effect of intrathecal electroporation and co-infusion on the occurrence of withdrawal signs in response to naloxone. A = vocalization in response to light touch with a piece of polyethylene tubing; B = spontaneous vocalization; C = abnormal posture; D = ejaculation; E = "wet dog head shakes"; F = escape attempts. Each group contained six rats. *Different from morphine 2 nmol/h on that day; P < 0.05. Please see the text for details of treatment groups.
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The time-course changes in tail withdrawal latency peaked at 30 min after intrathecal probe morphine administration. Thus, probe dose-response curves were generated on the basis of the % MPE at 30 min after probe dose administration. The curve in rats treated previously with 2 nmol/h intrathecal morphine infusions displayed a significant right shift (AD50 of M2, 124.7 nmol; 95% CI, 57–290 nmol), so the AD50 was increased by a factor of 14.5 as compared with saline-infused rats (AD50 of M0, 8.6 nmol; 95% CI, 4.7–15.57 nmol). This indicates that in morphine-tolerant animals, the potency of the probe dose of spinal morphine is considerably reduced (Fig. 3).
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Figure 3. Tail withdrawal latency and percentage of maximal possible effect (% MPE) 30 min after intrathecal probe morphine administered on Day 8 after a 7-day continuous intrathecal morphine infusion. Each point represents mean ± SD (n = 6). Please see the text for details of treatment groups.
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Morphine-infused rats that received 200 V of intrathecal electric stimulation showed a significant left shift in their dose-response curve (AD50 of M2 + V200, 13.6 nmol; 95% CI, 3.7–28.3 nmol) as measured by % MPE, such that the AD50 was significantly decreased by a factor of 9.2 as compared with that of morphine-infused rats (AD50 of M2, 124.7 nmol; 95% CI, 57–290 nmol).
In the spinal microdialysis study, baseline concentrations of all amino acids except GABA were detectable in all animals tested, with no significant differences among experimental groups (data not shown). In the rats that received 0 or 20 V of electric stimulation (M0, M0 + V20, M2, and M2 + V20), there were no significant differences in the concentrations of glutamate, aspartate, glycine, or citrulline for Days 0–7.
In the rats that received 200 V of electric stimulation (M0 + V200 and M2 + V200), there was no significant difference in the time-course changes in CSF amino acids between groups. In morphine-infused rats that received 200 V of intrathecal electric stimulation, there was a significant and prolonged increase in the concentration of glycine, increasing to 188% ± 39% of baseline values on Day 1, 193% ± 27% on Day 2 (Fig. 4B), and 286% ± 67% and 147% ± 25% on Days 3 (Fig. 4C) and 7, respectively. Concentrations of glutamate were modestly increased (P < 0.05) for the first 3 days (Fig. 4, B and C). The increase was detected at Days 1 (131% ± 49%), 2 (129% ± 38%), and 3 (134% ± 41%). Aspartate levels were slightly increased on Day 1 (125% ± 37%; P < 0.05) and then gradually normalized, with no significant change at Day 2 (116% ± 32%). Citrulline concentrations were unchanged (Fig. 4, B and C).
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Figure 4. Concentrations of glutamate, aspartate, glycine, and citrulline in spinal microdialysate samples expressed as percentage of baseline release (data are presented as means ± SD; n = 6; *P < 0.05). A, Time course of concentrations of cerebrospinal fluid (CSF) amino acids in morphine-infused rats without intrathecal electric stimulation (M2). B, Dose effects of electric stimulation on release of all amino acids on Day 2 in intrathecal morphine-infused rats. C, Effects of intrathecal electric stimulation in morphine-infused rats (M2, M2 + V20, and M2 + V200) on Day 3. D, Effects of electric stimulation in rats receiving 2 mg/kg of naloxone-induced CSF amino acid (AA) changes. Please see the text for details of treatment groups.
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One hour after naloxone treatment on Day 7 after saline infusion (M0, M0 + V20, and M0 + V200), there were no significant differences in the concentrations of CSF amino acids for the time points of pre- and postnaloxone treatment. One hour after naloxone treatment on Day 7 after morphine infusion (M2), there was a significant increase in glutamate (196% ± 55%; P < 0.05) but no significant change in the glycine level (Fig. 4D).
In comparison with morphine-infused-only animals (M2), morphine-tolerant rats receiving 200 V of electric stimulation (M2 + V200) significantly suppressed the increase of glutamate compared with those that received no treatment (M2; P < 0.05). No similar effect was seen in the group receiving 20 V of electric stimulation (M2 + V20) (Fig. 4D).
Light microscopy analysis showed no evidence of pathologic changes. All neuronal pools, including large A-motor neurons and small- and medium-sized interneurons, showed fully preserved nuclei and nucleoli, with no detectable changes in neuropils (data not shown).
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Discussion
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Intrathecal electric stimulation with this specially designed probe increased intrathecal morphine sensitivity and attenuated the development of rats’ tolerance to morphine, as shown in the challenge test. It also alleviated naloxone-precipitated morphine withdrawal symptoms, like previous techniques (14,15).
The biochemical basis by which this technique ameliorates morphine tolerance and withdrawal symptoms is unknown. We examined spinal neurochemical changes in both morphine-tolerant and electrically-stimulated rats. The morphine-tolerant rats did not show spinal neurochemical changes; however, in the rats receiving electrical stimulation, increased glycine levels were related to analgesia, and excitatory acid release was suppressed in the naloxone challenge test.
According to our previous work (11,12), this novel method stimulated the dorsal spinal cord (approximately 500 µm deep) and the anterior and posterior spinal nerve roots. In Laminae I and II of the spinal dorsal horn, there are key central nervous system areas in which pain-related, or nociceptive, information carried by sensory afferents is integrated and relayed to higher brain structures. Thus, the control of excitability of Laminae I–II neurons has a major effect on pain perception. Glycine and GABA are the main transmitters responsible for inhibitory control in this region and therefore act in nociception (16).
Immunocytochemical studies show colocalization of GABA and glycine, as well as their respective receptors, in the dorsal horn (17), and most glycinergic neurons in this region are also immunoreactive for GABA (18). GABA and glycine are co-released from individual vesicles at some synapses and from interneurons in the immature ventral horn to activate both postsynaptic GABAA receptors and glycine receptors simultaneously (19).
GABAA agonists, as well as GABAB agonists, potentiate the somatic and visceral antinociceptive effects produced by morphine. Systemically administered, muscimol or baclofen potentiate morphine-induced antinociception (20). Benzodiazepines, which mainly act on GABAA receptors, enhance opioid-induced antinociception at the spinal level (21). However, there is immunoreactivity for GABA and enkephalin in superficial dorsal horn neurons, and GABAergic neurons are connected to primary afferents in the dorsal horn (22). Some µ-opioid receptor-expressing neurons in the superficial dorsal horn are postsynaptic to GABAergic axon terminals (23). This suggests an antinociceptive interaction of GABA receptors with opioid receptors. Thus, intrathecal electric stimulation may potentiate the antinociceptive effect of morphine via increased levels of GABA and glycine.
Glutamate receptors are implicated in the genesis of opioid tolerance and dependence. In morphine-infused, but not control, rats, naloxone evoked an immediate increase in L-glutamate release, and this was significantly correlated with behavioral indices of withdrawal intensity (24). Electrical stimulation suppressed the naloxone-induced increases in spinal L-glutamate and taurine release and behavioral signs of withdrawal in spinal morphine-infused rats. These findings suggest a GABA- and glycine-mediated suppression of glutamate release (25). However, further investigation is warranted of the exact mechanisms by which intrathecal electric stimulation alters morphine antinociception.
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Acknowledgments
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Supported by National Science Council Grants NSC91-2314-B-182A-067, NSC91-2314-B-110-010, and CMRP8020.
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Accepted for publication January 24, 2003.
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Abstract
Introduction
