Gabapentin and NMDA receptor antagonists interacts synergistically to alleviate allodynia in two rat models of neuropathic pain

2.1 Photochemically-induced ischemic spinal cord injury

Ischemic spinal cord injury was produced in female SD rat weighing 200 g according to methods described previously [13]. In brief, rats were anesthetized with chloral hydrate (300 mg/kg, i.p.) and a midline incision was made on the skin overlying vertebral segments T 12-L 1. The animals were positioned beneath an argon laser beam and irradiated for 10 min with the beam directed towards vertebral segment T 12 or T 13 (spinal segments L 3–5). Immediately prior to and 5 min after the start of the irradiation, erythrosin B (Red N°3, Aldrich-Chemie, Steinheim, Germany) dissolved in 0.9% saline was injected intravenously through the tail vein at a dose of 32.5 mg/kg. A tunable argon ion laser (Innova model 70, Coherent Laser Product Division, Palo Alto, CA, USA) operating at 514 nm was used. The average beam output power was 160 mW. The beam covers the entire width of the vertebra and the length is approximately 1–2 mm. After irradiation, the wound was closed in layers and the rats were allowed to recover. Bladder was empted manually for 1 week.

2.2 Assessment of mechanical and cold sensitivity after spinal cord injury

The behavioral assessments were conducted blindly as the groups of drugs administered. Vocalization thresholds to graded mechanical touch/pressure were tested with calibrated von Frey hairs (ranging from 0.04 to 0.2155 mN, Stoelting, Chicago, IL, USA). During testing the rats were gently restrained in a standing position and the von Frey hair was pushed onto the skin until the filament became bent. The frequency of stimulation was about 1/s and at each intensity, the stimuli were applied 5–10 times. The intensity of stimulation which induced consistent vocalization (>75% response rate) was considered as pain threshold.

The response of rats to brushing stimulation was tested with the blunt point of a pencil gently stroking the skin on the trunk in a rostro-caudal direction. The frequency of the stimulation was about 1 Hz and responses were graded with a score of 0=no observable response; 1=transient vocalization and moderate effort to avoid probe; 2=consistent vocalization and aversive reactions and 3=sustained and prolonged vocalization and aggressive behaviors. Normal rats exhibited no reactions to such brush stimuli (score 0).

Responses to cold was tested with ethyl chloride spray applied to the shaved allodynic skin area. The response was graded with a score of 0=no observable response; 1=localized response (skin twitch and contraction), no vocalization; 2=transient vocalization, moderate struggle and 3=sustained vocalization and aggression. Normal rats usually had response score of 0 or 1.

2.3 Photochemically-induced sciatic nerve injury

Male SD rats were anesthetized by chloral hydrate (300 mg/kg, i.p.) and the left sciatic nerve was exposed. The nerve trunk was gently dissected free from the surrounding tissue over a distance of about 1 cm proximal to trifurcation. The exposed nerve was irradiated with an argon ion laser for 2 min. The irradiation was performed with a knife-edged beam across the nerve. Aluminum foil was placed under the nerve to isolate the surrounding tissue and to reflect light. Just before the irradiation, erythrosin B (Aldrich, USA 32.5 mg/kg dissolved in 0.9% saline) was injected i.v. via the tail vein. After the surgery the wounds were closed in layers and the rats were returned to the cages for subsequent behavioral tests.

2.4 Assessment of mechanical sensitivity after sciatic nerve injury

The behavioral assessments were conducted blindly as the groups of drugs administered. To test of sensitivity to mechanical stimulation, the rats were placed in plastic cages with a metal mesh floor. The plantar surface of the hind paws was stimulated with a set of calibrated von Frey hairs (ranging from 0.04 to 0.2155 mN, Stoelting, Chicago, IL, USA) with increasing force until the animal withdrew the limb. Each monofilament was applied 5 times. The withdrawal threshold was taken as the force at which the animal withdrew the paw from at least three out of five consecutive stimuli.

2.5 Drugs and statistics

Gabapentin, dextromethorphan and MK-801 were obtained from Research Biochem Inc. (Natick, MA, USA) and dissolved in physiological saline. All drugs were injected i.p. in a volume of 0.1 mL/kg. The behavioral assessments were conducted blindly. Data are expressed as median±median absolute deviation (MAD) and analyzed with Friedman one way analysis of variance for repeated measurements and Wilcoxon signed-ranks test.

3.1 Spinally injured rats

As previously described, some spinally injured rats developed allodynia-like behavior manifested as reduction in vocalization threshold to mechanical touch stimulation applied by the von-Frey hairs or by brush and as increased response to cold stimulation applied by ethyl chloride spray. And saline treatment has no effect on either mechanical or cold sensitivity on our neuropathic pain models [13], [14], [15].

I.p. dextromethorphan or gabapentin did not alleviate allodynia-like behaviors at the doses up to 20 or 30 mg/kg, respectively (Figs. 1–3). Further increasing the dose of dextromethorphan or gabapentin produced numerous side effects, including sedation and motor impairment for gabapentin and hyperactivity for dextromethorphan. Combining gabapentin (7.5, 15 or 30 mg/kg) with low doses of dextromethorphan (5 or 10 mg/kg) significantly increased vocalization threshold to von-Frey hair stimulation (Fig. 1), reduced and normalized increased response to brush (Fig. 2) or cold (Fig. 3) stimulation. The anti-allodynic effect of dextromethorphan and gabapentin is long-lasting, but reversible. At doses used, gabapentin, dextromethorphan or combination did not produce observable side effects, such as sedation, motor impairments or hyperactivity.

Fig. 1:

Effects of gabapentin at 30 mg/kg, dextromethorphan at 20 mg/kg or combination of the two compounds on vocalization threshold to von-Frey hair stimulation in spinally injured rats. The data is expressed as median±MAD (median absolute deviation) and 6–8 rats were included in each group. *=p<0.05 compared to time 0 with Wilcoxon signed-rank test. Friedman ANOVA with repeated measures indicated a significant general difference for the three drug combination groups (p<0.01).

Fig. 2:

Effect of gabapentin at 30 mg/kg, dextromethorphan at 20 mg/kg or combination of the two compounds on responses of spinally injured rats to brushing stimulation. The responses were graded and number of rats exhibiting different level of responses were shown. *=p<0.05 compared to time 0 with Wilcoxon signed-ranks test.

Fig. 3:

Effect of gabapentin at 30 mg/kg, dextromethorphan at 20 mg/kg or combination of the two compounds on responses of spinally injured rats to cold stimulation. The responses were graded and number of rats exhibiting different level of responses were shown. *=p<0.05 compared to time 0 with Wilcoxon signed-ranks test.

I.p. MK-801 at 0.05 or 0.1 mg/kg also did not affect mechanical allodynia-like behavior in spinally injured rats whereas combination of small doses (0.01 or 0.05 mg/kg) of MK-801 with 15 mg/kg gabapentin again significantly increased vocalization threshold to von-Frey stimulation (Fig. 4). The cold allodynia was also similarly reduced (not shown).

Fig. 4:

Effect of MK-801 at 0.05 or 0.1 mg/kg or combination with MK with gabapentin 15 mg/kg on vocalization threshold of spinally injured rats to von Frey stimulation. The data is shown as median±MAD (median absolute deviation) and *=p<0.05 compared to time 0 with Wilcoxon signed-ranks test. Friedman ANOVA with repeated measures indicated a significant general difference for the two drug combination groups (p<0.01).

3.2 Sciatic nerve injury

Rats subjected to ischemically-induced sciatic nerve injury developed mechanical hypersensitivity seen as bilaterally decreased paw withdrawal threshold to von-Frey hair stimulation which peaked at 1–2 weeks when the experiments were conducted. The mechanical hypersensitivity is more severe on the ipsilatera side to the irradiation than the contralateral side and the data from the ipsilateral were presented. I.p. dextormethorphan or gabapentin did not affect mechanical allodynia in sciatic nerve injured rats in the present experiments as doses up to 40 or 100 mg/kg despite the presence of side effects (Fig. 5). In contrast, combining gabapentin 30 mg/kg with 20 mg/kg dextromethorphan significantly increased paw withdrawal threshold to von-Frey stimulation in nerve injured rats (Fig. 5).

Fig. 5:

Effects of gabapentin at 100 mg/kg, dextromethorphan at 40 mg/kg or combination of dextromethorphan 20 mg/kg and gabapentin 30 mg/kg on paw withdrawal threshold after partial sciatic nerve injury. The data is expressed as median±MAD (median absolute deviation) and 6–8 rats were included in each group. *=p<0.05 compared to time 0 with Wilcoxon signed-rank test. Friedman ANOVA with repeated measures indicated a significant general difference for the drug combination groups (p<0.01).