The Interaction Between Gamma-Aminobutyric Acid Agonists... : Anesthesia & Analgesia (original) (raw)
In animals, γ-aminobutyric acid (GABA) receptor agonists (1,2) and voltage-dependent calcium channel blockers (3,4) have antinociceptive effects at the spinal level. There are data suggesting that the two types of drugs may interact in the central nervous system (5–7). GABAB receptors have been considered to be connected with voltage-dependent calcium channels via the G protein Go (5). Calcium ion modulated GABAA receptor function in rat cerebral cortical neurons (6). GABA, acting at the GABAA receptor, induced calcium ion entry to rat embryonic dorsal horn neurons through high-threshold voltage-dependent calcium channels (7).
Visceral antinociceptive effects of the two types of drugs have also been studied. Systemic and spinal GABA agonists have visceral antinociceptive effects in rats (2,8,9). Spinal L-type calcium channel blockers have also been reported to have a visceral antinociceptive effect in animal studies in which the writhing test (10,11) and the colorectal distension (CD) test (4) were used. In contrast, clinical reports have described a successful use of intrathecal baclofen (BAC), a GABAB agonist, for the treatment of neuropathic and central pain (12,13) and suggested that BAC was useful for those types of pain. The effect on pain related to visceral origin has not been examined in a clinical situation. Although intrathecal or epidural L-type calcium channel blockers have been used for cancer pain with morphine (14) and for postoperative pain with bupivacaine (15), the visceral antinociceptive effects of GABA agonists with voltage dependent calcium channel blockers have not been studied.
In the present study, accordingly, we intrathecally administered a GABAA (muscimol, MUS) or GABAB (BAC) agonist and/or an L-type calcium channel blocker (diltiazem, DIL) in rats to determine whether the two types of drugs would potentiate each other on the visceral antinociception at the level of spinal cord.
Methods
Male Sprague-Dawley rats weighing 250~400 g were maintained in a climate-controlled room on a 12:12 h light-dark cycle (lights on at 08:00) with free access to food and water. The protocol for this experiment was approved by the Animal Research and Use Committee of Shimane University School of Medicine.
To reduce the influence of handling on nociceptive responses, all animals were handled and trained in the test situation at least 2 times before intrathecal catheterization. Under pentobarbital (40 mg/kg) anesthesia, intrathecal catheters (double-stretched PE-10 connected to normal PE-10 and to PE 20) were implanted in the rostral direction from the level of L4-5 intervertebral space. After catheterization, at least 5 days was allowed for recovery before the experiment. Rats that had motor deficits as a result of catheter placement, infection, or other health problems were excluded.
For intrathecal administration, the drugs were given in a volume of 10 μL, followed by saline 10 μL to flush the catheter. The investigators were blinded to the solutions injected. Groups of 7 rats each received intrathecal administration of saline, DIL (100 μg), MUS (0.1 or 1 μg), or BAC (0.01 or 0.1 μg), or combinations of DIL and either MUS or BAC. Dosage of drugs administered was determined according to the results of our previous studies (2,4). Some rats were tested on multiple days (not more than 3) but did not receive the same drug more than once, and only those that recovered completely were used. Animals were given at least 2 days rest period between drug administrations to exclude any possibility of drug interaction.
The CD test was performed to measure noxious visceral stimuli by using an 8 cm long, flexible, latex balloon. The balloon consisted of two parts: a large proximal stimulating balloon and a small distal sensing balloon. Both stimulating and sensing balloon pressures were continuously monitored via inline pressure transducers and recorded. The balloons were inserted intra-anally into the colon and rectum under light halothane anesthesia. Animals were tested while awake after a minimum 20-min recovery from halothane anesthesia. Pressure within the intracolonic-stimulating balloon was steadily increased at a rate of 2.5 mm Hg/s beginning at 0 mm Hg until a rapid increase in pressure of the sensing balloon was observed. The increase in pressure of the sensing balloon reflects abdominal musculature contractions. The pressure in the stimulating balloon at which the abdominal musculature contractions were triggered was defined as the threshold response for the nociception. A cutoff distension pressure of 60 mm Hg was used to prevent tissue damage.
Motor function after intrathecal injection was assessed by bilaterally grading the motor block in the upper and lower limbs as follows: 0 = none; 1 = partially blocked; and 2 = completely blocked. Motor blockade was graded as none when the rat had no visible limb weakness and normal gait; as partially blocked when the limb was able to move but not able to support the normal posture; and as completely blocked when the limb was flaccid, with no detectable resistance to extension of the limb. The normal score was 0, and the score with bilateral complete blockade in the upper or lower limbs was 2 + 2 = 4.
Nociceptive tests and evaluation of motor function were performed before and 5, 10, 15, 20, 30, 60, 90, and 120 min after intrathecal injection. At the end of the experiment, each rat received 2% lidocaine (10 μL) through the catheter. Data obtained from rats in which paraplegia after lidocaine was not observed were excluded from the data analysis.
The antinociceptive effects were evaluated by transforming CD threshold to the percent of maximum possible effect (%MPE), calculated as (postdrug value − baseline value)/(cutoff value − baseline value) × 100%. Motor function scores are presented as median and tenth and ninetieth percentiles, and the other data as mean ± sem. %MPE after the drug administration were compared with the saline control or other drug administration groups using an analysis of variance (ANOVA) for repeated measures, followed by Fisher’s PLSD test. Comparison between the groups was accomplished by testing Kruskal-Wallis analysis of variance by ranks to assess motor function. Individual comparisons were performed with the Mann-Whitney _U_-test. Differences were considered to be significant at P < 0.05.
Results
Intrathecally-administered saline produced no change in the CD test (Figs. 1 and 2). Intrathecal MUS 0.1 μg or DIL 100 μg did not affect CD threshold, but the combination of the two significantly increased the value with a peak effect of 26%MPE at 5 min. Intrathecal MUS 1 μg slightly increased CD threshold with a peak effect at 5 min after the administration (20%MPE). When combined with DIL 100 μg, the increase in CD threshold was significantly larger compared with that after MUS 1 μg alone (Fig. 1).
Time-course effects on percent maximum possible effect (%MPE) in colorectal distension (CD) test after intrathecal administration of saline (SAL), diltiazem 100 μg (DIL 100), muscimol 0.1 μg (MUS 0.1), muscimol 1 μg (MUS 1), muscimol 0.1 μg + diltiazem 100 μg (MUS 0.1+ DIL 100), or muscimol 1 μg + diltiazem 100 μg (MUS 1+ DIL 100); n = 7 for each group. Data are presented as mean ± sem. *P < 0.05 compared with SAL; #P < 0.05 compared with MUS 0.1, MUS 1, or DIL 100 alone.
Time-course effects on percent maximum possible effect (%MPE) in colorectal distension (CD) test after intrathecal administration of saline (SAL), diltiazem 100 μg (DIL 100), baclofen 0.01 μg (BAC 0.01), baclofen 0.1 μg (BAC 0.1), baclofen 0.01 μg + diltiazem 100 μg (BAC 0.01+ DIL 100), or baclofen 0.1 μg + diltiazem 100 μg (BAC 0.1 + DIL 100); n = 7 for each group. Data are presented as mean ± sem. *P < 0.05 compared with SAL; #P < 0.05 compared with BAC 0.01 or BAC 0.1, or DIL 100 alone.
Figure 2 shows the effects of the combination of BAC and DIL. Intrathecally-administered BAC 0.01 μg showed no increase but 0.1 μg a slight increase in CD threshold. The combination of BAC 0.01 μg and DIL 100 μg showed no significant change in CD test. The combination of BAC 0.1 μg and DIL 100 μg produced a significant increase in CD threshold that was significantly larger compared with the increase after BAC alone at 5 min after the administration. A peak effect was observed 5 min after the administration (48%MPE) and the increase in the effect was maintained for 20 min.
No rats showed any effects on motor function after intrathecal injection of BAC or DIL. MUS 0.1 μg alone or in combination with DIL did not affect the motor function score during the study. In contrast, MUS 1 μg with or without DIL produced apparent motor paralysis in the lower limbs, but the score did not differ between MUS alone and MUS administered with DIL (Table 1). Peak effects on motor function were observed at 30 and 60 min after the administration of MUS and the combination of MUS and DIL, respectively.
Motor Function Score
Discussion
The present study demonstrated that the visceral antinociceptive effects produced by intrathecally-administered MUS and BAC were potentiated by co-administered DIL, which alone did not show any effects on antinociception. These results suggest that both the activation of GABAA or GABAB receptors and the inhibition of calcium influx suppress visceral nociceptive transmission at the spinal level.
GABA receptor agonists and L-type calcium channel blockers have been used in humans. For example, systemic GABAA agonists were used in patients with chronic cancer pain only to show insufficient analgesic effect (16). BAC has been used clinically for decades as a muscle relaxant or an antispastic drug (17) and has been tried for relieving intractable neuropathic pain (12,13). However, an analgesic effect of BAC on visceral pain has not been reported. Nimodipine, an L-type calcium channel blocker, has been administered epidurally with morphine for cancer pain (14). Epidural verapamil with bupivacaine was reported to reduce analgesic consumption after lower abdominal surgery (15). Although the results of animal studies have suggested visceral antinociception of L-type calcium channel blockers (4,10,11), few researchers have administered them alone for the treatment of visceral pain in humans.
The control of visceral pain is clinically important because visceral pain plays a major part in cancer and postoperative pain. The present results demonstrated that the combination of GABA agonists and L-type calcium channel blockers can reduce visceral pain. Furthermore, although both GABA agonists and L-type calcium channel blockers alone had motor blocking effect, the coadministration of the two types of drugs did not aggravate motor dysfunction. Motor dysfunction is a major side effect and limits ambulation. Preserving the ability to ambulate is desirable for patients with postoperative and cancer pain. Thus, the present results showing better analgesia without an increase in motor dysfunction suggest that intrathecal and epidural coadministrations of GABA agonists and DIL are a possible clinical choice in the future.
However, in the present experiment, other possible side effects were not evaluated. GABAA agonist has been associated with minor side effects, including sedation, dizziness, euphoria, nausea, and blurred vision (16). Neurologic side effects, such as hallucinations and seizures, have been reported after precipitous withdrawal of BAC (18). Systemic L-type calcium channel blockers have cardiovascular effects, and their spinal administration may produce hypotension and/or arrhythmia. However, these side effects, including motor weakness, are dose dependent. Thus, it is unlikely that the combination of the two types of drugs at small doses increases the degree of side effects.
The influence of motor block on a nociceptive test should be considered in the interpretation of results because abdominal musculature contractions may be obscured by muscle weakness. In the present study, motor block was restricted to the lower limbs. The peak effects in the CD test occurred at 5 min when motor paralysis was not apparent, and peak effects on motor function occurred at 30 and 60 min after the administration of MUS and the combination of MUS and DIL, respectively. Thus, it is unlikely that motor dysfunction affected the present results in CD tests.
The different distribution of receptors may explain less inhibition of motor activity by BAC. Although the concentrations of both GABAA and GABAB receptor binding sites are similar in the ventral horn, the latter exists more than the former in the substantia gelatinosa (19). The reasons for different potentiation of antinociception and motor dysfunction are also speculative. Different types of nerves should differ in the type and/or the number of receptors and channels (20). In addition, the dorsal roots have larger surface for drug penetration than the ventral nerve roots (21).
In conclusion, intrathecal administration of DIL in combination with a GABA agonist, MUS or BAC, potentiated the GABA agonists-induced antinociception and did not increase motor paralysis. Although the mechanism of interaction between GABA agonists and L-type calcium channel blockers on antinociception in the spinal cord remains to be elucidated, the present results indicate that the coadministration of the two types of drugs may be clinically useful.
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