The experiments were conducted in male Sprague-Dawley rats (weight 200-250 g) in a study performed using a protocol approved by the animal use and care committee. The animals were housed individually in a temperature-controlled vivarium and allowed to acclimate for 3 days in a 12/12 hr light/dark cycle.

For intrathecal drug administration, the rats were chronically implanted with catheters as previously described (20). Briefly, under sevoflurane anesthesia, the rats were placed in a stereotaxic head holder and the occipital muscles were separated from their attachment points, retracting the muscles caudally to expose the cisternal membrane at the base of the skull. Intrathecal catheters (PE-10 polyethylene tubing, Becton Dickinson, Sparks, MD, USA) were passed caudally from the cisterna magna to the spinal cord level of lumbar enlargement and then externalized through the skin. Proper intrathecal location was confirmed by a temporary motor block of both hind-limbs after the injection of 2% lidocaine 10 µL, followed by saline. Only animals with no evidence of neurologic deficit after the operation were studied. At least a 5 day recovery period was allowed before the animals were used in experiments.

Morphine sulfate (molecular weight, 668.76), lamotrigine (molecular weight, 256.09), and dimethyl sulfoxide (DMSO, minimum 99.5%) were purchased from Sigma, St. Louis, MO, USA. Lamotrigine was dissolved in DMSO, and morphine in 0.9% saline. The doses for intrathecal lamotrigine were selected according to a previous study (14, 21). All drugs were administered intrathecally with a microinjection syringe (Microliter™ #702, Hamilton Co., Reno, NV, USA) over a 60 sec interval in a volume of 10 µL, followed by a 10 µL saline flush. Investigators were blind to the administered drug.

Thermal nociception was assessed by measuring the latency of paw withdrawal in response to a radiant heat source (22). Rats were housed individually in Plexiglas chambers on an elevated glass platform, under which a radiant heat source (Ugo Basile, Milan, Italy) was applied to the plantar surface of the hind-paw through a glass plate maintained at 30℃. The heat source was turned off when the rat lifted its foot, and the time from the onset of radiant heat application to hind-paw withdrawal, defined as paw withdrawal latency (PWL), was recorded. The heat was maintained at a constant intensity and resulted in a stable PWL of approximately 10-12 sec in normal rats. A 20 sec cut-off avoided tissue damage. PWL was measured three times at 5 min intervals and the average values were calculated. The measured PWL was converted to a percentage of maximum possible effect (%MPE) according to the formula: %MPE = ([post-drug value-baseline value]/[cut-off value-baseline value]) × 100%.

Single intrathecal doses of 5 µg of morphine, 200 µg of lamotrigine, and a combination of both drugs were studied (n = 6 per subgroup). Measurements were taken before and 15, 30, 45, 60, 90, 120, and 180 min after an intrathecal dose of the drug(s).

Rats in the morphine group (M1-7 group, n = 6) received intrathecal injections of 15 µg of morphine once daily for 7 days. This dose was previously shown to produce tolerance over 7 days following initial maximal antinociception (23). To evaluate the effects of lamotrigine on the development of morphine tolerance, a dose of 200 µg was intrathecally co-injected with 15 µg of morphine once daily for 7 days (L1-7 group, n = 6). The offset of lamotrigine effect on morphine tolerance was determined by co-injecting the two drugs on days 1-3 followed by the daily injection of morphine alone on days 4-7 (L1-3 group, n = 6). Behavioral testing was performed before and 30 min after the drug administration.

Intrathecal morphine (15 µg) was given once daily for 4 days to induce tolerance. On the following 3 days, intrathecal lamotrigine (200 µg) was co-administered with the daily dose of morphine (L5-7 group, n = 6).

To evaluate microglial and astrocytic activation in the spinal cord after the development of morphine tolerance, the cells were stained with the microglial marker OX-42 and glial fibrillary acidic protein (GFAP), respectively. After the last behavioral assessment, the rats were deeply anesthetized with sevoflurane and perfused intracardially with heparinized normal saline, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS) on day 8. The spinal cord around lumbar vertebrae L5 and L6 was removed and then post-fixed in the same fixative at room temperature for 4 hr. All specimens were cryoprotected in 30% (w/v) sucrose. The spinal cords were cut transversely using a cryostat into 20 µm floating sections, washed first in PBS and then 2 × 10 min in PBS containing 0.3% Triton X-100 (PBST), and finally treated with 0.3% hydrogen peroxide in PBST for at least 15 min to exhaust endogenous hydrogen peroxidase. Non-specific binding was blocked by incubating the sections in 3% normal goat serum plus 2% bovine serum albumin in PBST for 1 hr. Next, the sections were incubated at room temperature overnight with primary mouse monoclonal antibody (anti-OX-42, 1 µg/mL; Serotec, Oxford, UK) in PBST plus 3% normal goat serum. On the following day, they were washed 3 × 10 min with PBST and then incubated with goat anti-mouse biotinylated secondary antibody (1:1,000; Chemicon, Temecula, CA, USA) for 1 hr at room temperature. Following three washes with PBST, the floating sections were incubated for 1 hr with a 1:160 dilution of an avidin-biotin horseradish peroxidase complex (ABC complex; Pierce, IL, USA) in PBST. Antigens were visualized by treating the stained sections with a 1:1 solution of ammonium nickel sulfate (30 mg/mL in 0.1 M sodium acetate, pH 6.0) and diaminobenzidine (4 mg/mL in PBS) in the presence of 0.01% hydrogen peroxide. The sections were then spread flat on slides, air-dried, rinsed with distilled water for 1 min, and dehydrated first through an ethanol gradient (70% once, 95% twice, and 100% twice, 1.5 min each) and then in xylene (2 × 3 min). The processed sections were cover slipped with Permount mounting medium (Merck, Darmstadt, Germany). Images were acquired with a DXM1200 digital camera connected to an Eclipse E800 light microscope (Nikon, Melville, NY, USA).

Glial responses were assessed in three randomly chosen sections prepared from the L5 spinal cord segments. The number of immunoreactive cells in the medial superficial dorsal horn (laminae I-III), where most of the positive cells were found after the treatments, were counted. The same microscopic field was photographed at a magnification of 100 × using a digital camera. The data were expressed as the number of positive cells/area/spinal section.

The behavioral data were expressed as the mean ± SEM. Statistical analyses were carried out using the one-way analysis of variance (ANOVA), comparing the treatment groups to the control group at the same time point, followed by a Tukey's test for multiple comparisons. The significance of the differences in the responses of the treatment groups compared to the respective preoperative baseline values was determined using one-way repeated-measures ANOVA followed by a Tukey's test for multiple comparisons. Statistical evaluation was performed with SigmaPlot® software version 11 (Systat Software, Inc., San Jose, CA, USA). P < 0.05 was considered statistically significant.

In this study design, 54 rats were used in total (Table 1). In the study 1, acute effects of lamotrigine on morphine antinociception were investigated in 24 rats (six rats per group × four groups). In the study 2, effects of lamotrigine on the development of morphine tolerance were analyzed in 24 rats (six rats per group × four groups). In the study 3, effects of lamotrigine on established morphine tolerance was analyzed in 6 rats (six rats per group × one group).

Submaximal doses of intrathecal morphine (5 µg) produced peak antinociceptve effect against thermal stimulation (55.4 ± 5.2 %MPE) 30 min after intrathecal administration of morphine (Fig. 1). A 200 µg intrathecal dose of lamotrigine alone likewise produced peak antinociceptive effect against thermal stimulation (32.0 ± 5.7 %MPE) 30 min after administration (Fig. 1). When the two drugs were injected together at these doses, an increase in antinociception (98.9 ± 1.3 %MPE) peaking 30 min later was recorded. The combination of morphine and lamotrigine resulted in significantly larger antinociceptive responses than morphine alone (P<0.001) (Fig. 1). All responses returned to baseline by 180 min after drug injection. Visual inspection of the treated animals revealed no signs of motor impairment. Intrathecally administered vehicle (10% DMSO) had no effect (data not shown).

In the M1-7 group, the administration of intrathecal morphine (15 µg) produced maximal antinociception against thermal stimulation on day 1 (99.7±4.2 %MPE), with the effect decreasing to baseline levels by day 7 (18.6±4.8 %MPE). In the L1-7 group, the co-administration of intrathecal morphine (15 µg) and intrathecal lamotrigine (200 µg) completely blocked the decrease in morphine's effect throughout the entire 7 day period (90.1±3.6 %MPE on day 7, Fig. 2). In a subsequent experiment (L1-3 group), in which lamotrigine was co-administered with morphine only on days 1-3, maximal antinociception with morphine was still present on day 4 (92.1±3.2 %MPE) but the effect decreased over days 5-7 (55.6±3.7 %MPE on day 7, Fig. 2).

In the L5-7 group, the chronic administration of morphine alone on days 1-4 resulted in a decrease in antinociception against thermal stimulation (54.7±4.5 %MPE on day 4, Fig. 2). However, the addition of lamotrigine on days 5-7 partially restored morphine's effect (68.7±5.1 %MPE on day 5, P=0.026 vs M1-7 group) such that antinociception on days 5-7 was significantly greater than obtained with morphine alone (P=0.042 on day 6, P=0.003 on day 7) (Fig. 2).

The immunoreactivity (IR) of OX-42 in the L5 spinal cord is described in the Fig. 3. In the control group, there were 31.5±2.3 OX-42-positive cells (under 200×magnification), whereas with once-daily intrathecal injections of 15 µg of morphine, OX-42 IR significantly increased to 257.7±13.3 cells (P<0.001 vs control group) after 7 days (M1-7 group). These findings implicate microglial activation in the effects of morphine. Following the co-administration of 200 µg of lamotrigine for 7 days (L1-7 group), there were significantly fewer stained cells (194.2±11.2, P=0.013 vs M1-7 group). When 200 µg of lamotrigine was coinjected with morphine on days 1-3 followed by daily morphine alone on days 4-7 (L1-3 group), there were 208.5±13.9 OX-42-positive cells. Conversely, the coadministration of 200 µg of lamotrigine and 15 µg of morphine on days 5-7 (L5-7 group) resulted in 168.3±10.4 OX-42-positive cells. In these three groups (L1-7, L1-3, and L5-7), although the numbers of OX-42-positive cells were significantly lower than those of the M1-7 group (P=0.013, P=0.045, P<0.001), they were significantly higher (P<0.001) than those of the control group.

The IR of GFAP in the L5 spinal cord is explained in the Fig. 4. In the control group, there were 39.3±3.3 GFAP-positive cells (under 200×magnification). However, with intrathecal injections of 15 µg of morphine once daily for 7 days (M1-7 group), GFAP IR significantly increased to 152.8±12.7 (P<0.001). These findings implicate astrocytic activation in the response to morphine. By contrast, following the co-administration of 200 µg of lamotrigine for 7 days (L1-7 group), the number of GFAP-positive cells significantly decreased to 110.3±8.7 (P=0.015 vs M1-7 group), respectively. When 200 µg of lamotrigine was coinjected with morphine for days 1-3 followed by daily morphine alone on days 4-7 (L1-3 group), the number of GFAP-positive cells was 94.5±7.4. In the group co-administered with 200 µg of lamotrigine with morphine on days 5-7 (L5-7 group), the corresponding cell number was 115.3±8.3. In these three groups (L1-7, L1-3, and L5-7), the numbers of GFAP-positive cells were significantly lower (P=0.015, P<0.001, P=0.044) than those of the M1-7 group, however, they were significantly higher (P<0.001) than the numbers of GFAP-positive cells of the control group.