The experiments were performed under a protocol approved by the Animal Care Committee. Male Sprague-Dawley rats (weight 160-180 g) were housed individually in a temperature-controlled (21 ± 1℃) vivarium and allowed to acclimate for three days in a 12/12-h day/night cycle.

To create the SNL model, a surgical procedure was performed according to the method described by Kim and Chung [1]. Under the enflurane anesthesia, the left L5 and L6 spinal nerves were isolated and ligated tightly with 6-0 black silk distal to the dorsal root ganglion and proximal to the formation of the sciatic nerve. The IT catheter was implanted if the rat showed a withdrawal threshold of 4.0 g or less by postoperative day 7, which indicated mechanical allodynia. The implantation of IT catheter was performed as previously described [7]. Under enflurane anesthesia, a polyethylene tube (PE-10) was passed 8.5 cm caudally from the cisterna magna to the rostral edge of the lumbar enlargement and externalized through the skin. Only animals without the evidence of neurologic deficit after the operation were included in this study. Rats were kept in individual vivaria and allowed to recover for 5 days after catheter implantation.

Behavioral testing was done at the same time during the day (9:00 AM to 4:00 PM). Rats were placed in individual plastic cages with wire mesh bottoms and allowed to acclimate for 20 min. The mechanical threshold was measured by applying a von Frey filament (Stoelting, Wood Dale, IL, USA) vertically to the midplantar surface of the hindpaw ipsilateral to the nerve ligation injury until a positive sign for pain behavior was elicited. According to the method described by Chaplan et al. [8], a series of eight calibrated von Frey filaments (0.41, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.10 g) was applied serially to the paw in ascending order of strength with sufficient force to cause gentle bending against the paw and held for six seconds. A sharply withdrawal or paw flinching was considered a positive response. The mechanical stimulus producing a 50% likelihood of withdrawal was determined by using the up-down method. Measurements were taken before and 15, 30, 60, 90, and 120 min after the IT administration of each drug. Side effects were simply evaluated by observing the presence of sedation and motor weakness. Severe sedation was defined as a significant decrease in spontaneous activity and a loss of the orienting reflex to light touch. Motor weakness was assessed by observing abnormal ambulation, abnormal weight bearing, or righting and stepping reflexes.

The following drugs were used in this study: neostigmine bromide (Research Biochemicals International [RBI], Natick, MA, USA), CHA (RBI), and pirenzepine (RBI). All drugs were dissolved in normal saline and were administered intrathecally with a microinjection syringe (Hamilton Co., Reno, NV, USA) over a 60 second interval in a volume of 10 µl, followed by a 10-µl flush.

The first series of experiments defined the dose-response curves of intrathecal administered neostigmine (0.3, 1, 3, and 10 µg) and CHA (0.03, 0.1, 0.3, 1, and 3 µg) from the mean percent maximal possible effect (%MPE) to establish the ED₅₀s of two drugs. In the second series of experiments, ED₅₀ fractions (1/2, 1/4, and 1/8) of each drug were administered concurrently to establish the ED₅₀ of neostigmine-CHA combinations. Thereafter, the drug interactions were evaluated by isobolographic and fractional analyses. In the third series of experiments, to identify a possible mechanism of the interaction between neostigmine and CHA, muscarinic antagonist pirenzepine 3 µg was administered intrathecally 10 min before the injection of both drugs.

To determine whether the drug interaction between neostigmine and CHA is additive or synergistic, an equal dose ratio isobolographic analysis was conducted by using the method of Tallarida and Murray [9]. The theoretical additive combination dose was calculated by the method described by Tallarida et al. [9,10]. The experimental ED₅₀ values were compared with the theoretical additive ED₅₀ values as defined by the theoretical additive line. The theoretical additive ED₅₀ point lies on a line connecting the ED₅₀ values of the individual drugs. The experimentally derived value that lies below and to the left of the theoretical additive line is considered to be synergistic, whereas value that lies above and to the right of the line demonstrates an antagonistic interaction.

Fractional analysis was performed to obtain a value for describing the magnitude of the drug interaction. A total fraction was calculated with the following formula: (ED₅₀ dose of neostigmine in combination / ED₅₀ value for neostigmine alone) + (ED₅₀ dose of CHA in combination / ED₅₀ value for CHA alone). A value near 1 implies an additive interaction, and value less than 1 indicates a synergistic interaction [11].

The peak drug effects were recorded and then used to calculate %MPE values. These %MPE values were plotted versus the log dose for dose response data. The withdrawal threshold data from von Frey filament testing were obtained as the actual threshold in grams and converted to %MPE values by the following formula: %MPE for anti-allodynia = ([post-drug threshold - baseline threshold] / [15 g - baseline threshold]) × 100. The cut-off value was defined as a stimulus intensity of 15 g for the mechanical threshold (i.e., %MPE = 100).

Data are presented as mean ± SEM or 95% confidence intervals (CIs). The ED₅₀ values, slopes, and 95% CIs were determined using a dose-response analysis. Variances and their 95% CIs for the theoretical additive ED₅₀ were calculated from the variances of each component administered alone. The difference between the theoretical additive point and the experimentally derived ED₅₀ was compared by a Student t-test. The effect of drugs on mechanical allodynia was tested by one-way analysis of variance for repeated measures followed by Dunn's post hoc test. The difference between the agonistic effect and the antagonistic effect was tested by Mann-Whitney Rank Sum test. P value < 0.05 was considered statistically significant.

After spinal nerve ligation, all rats displayed a significant decrease in mechanical threshold (from 15 g to the range of 1 to 4 g) necessary to evoke a brisk withdrawal response in the injured hindpaw in response to von Frey filament stimulation.

IT administration of neostigmine, CHA, and their combination produced a dose-dependent anti-allodynic effect (Fig. 1). The maximal anti-allodynia effects occurred within 30 min, and then decreased gradually to baseline with the passage of time. The patterns of time-effect course were similar in all groups (data not shown).

The ED₅₀ values and slopes of neostigmine, CHA, and their combination are described in Table 1. The dose-response curve of the combination group was shifted to the left and steeper compared drug alone (Fig. 1). Isobolographic analysis indicated a synergistic interaction between neostigmine and CHA (Fig. 2). The experimentally determined mixture ED₅₀ (± SEM) was 0.07 µg (±0.016) for neostigmine and 0.02 µg (±0.004) for CHA. The theoretical additive ED₅₀ was calculated to be 0.18 µg (±0.04) for neostigmine and 0.05 µg (±0.01) for CHA. The experimental value of neostigmine-CHA combination was significantly less than the calculated additive value (P < 0.05). The total fraction for the neostigmine-CHA combination was 0.39, indicating a synergistic interaction (Table 2).

Pretreatment with the muscarinic M1 antagonist pirenzepine significantly reduced the anti-allodynic effect of IT neostigmine-CHA combination (P < 0.05, Fig. 3).

Some rats in all groups displayed a mild to moderate sedation and motor weakness, but no severe sedation or motor weakness. There was no apparent increase in the incidence and severity of side effects in the combination group (Table 3). After IT administration of either 10 µg of neostigmine or 3 µg of CHA, a moderate motor weakness was observed in two rats (one in the neostigmine group and one in the CHA group).