Evidence that pregabalin reduces neuropathic pain by inhibiting the spinal release of glutamate

Abstract

Pregabalin is an anti-convulsant that successfully treats many neuropathic pain syndromes, although the mechanism of its anti-hyperalgesic action remains elusive. This study aims to help delineate pregabalin's anti-hyperalgesic mechanisms. We assessed the effectiveness of pregabalin at decreasing mechanical and cold hypersensitivity induced in a rat model of neuropathic pain. Thus, we compared the effectiveness of pre- or post-treatment with systemic or intrathecal (i.t.) pregabalin at reducing the development and maintenance of the neuropathic pain symptoms. Pregabalin successfully decreased mechanical and cold hypersensitivity, as a pre-treatment, but was less effective at suppressing cold hypersensitivity when administered as a post-treatment. Furthermore, both i.t. and systemic administration of pregabalin were effective in reducing the behavioral hypersensitivity, with the exception of systemic post-treatment on cold hypersensitivity. We also examined pregabalin's effects at inhibiting hind paw formalin-induced nociception in naïve rats and formalin-induced release of excitatory amino acids in the spinal cord dorsal horn (SCDH) both in naïve rats and in rats with neuropathic pain. Pregabalin dose-dependently reduced nociceptive scores in the formalin test. We also present the first evidence that pregabalin reduces the formalin-induced release of glutamate in SCDH. Furthermore, i.t. pregabalin reduces the enhanced noxious stimulus-induced spinal release of glutamate seen in neuropathic rats. These data suggest that pregabalin reduces neuropathic pain symptoms by inhibiting the release of glutamate in the SCDH.

Fig. 1

Effect of intrathecal (i.t.) pregabalin pre-treatment on mechanical (A) and cold (B) sensitivity in rats after chronic constriction injury (CCI) of sciatic nerve. For von Frey thresholds, ANOVA indicated significant main effects of i.t. pregabalin pretreatment (F(3,24) = 10.0, p > 0.001), time (F(3,72) = 14.4, p < 0.001, and a significant treatment X time interaction (F(9,72) = 2.53, p < 0.05). For cold water frequencies, ANOVA indicated a significant main effects of i.t. pregabalin pretreatment (F(3,24) = 4.39, p < 0.05) and time (F(3,72) = 13.9, p < 0.001, and a non-significant treatment X time interaction (F(9,72) = 1.80, p > 0.05). Post hoc comparisons revealed that compared to vehicle (n=8) there was a significant reduction in mechanical hypersensitivity (A) with 30 (n=6) and 100 (n=6) μg i.t. pregabalin pretreatment on days 4, 8 and 12, and 10 μg (n=7) on day 8 after CCI (*p < 0.05), and cold hypersensitivity (B) was significantly reduced by 100 μg pregabalin on days 8 and 12 after CCI (*p < 0.05) as compared to vehicle (n’s same as in A).

Fig. 2

Effect of systemic (i.p.) pregabalin pretreatment on mechanical (A) and cold (B) sensitivity in rats after chronic constriction injury (CCI) of sciatic nerve. For von Frey thresholds, ANOVA indicated a significant main effects of i.p. pregabalin pretreatment (F(3,31) = 4.17, p < 0.05) and time (F(3,93) = 31.6, p < 0.001, as well as a significant treatment X time interaction (F(9,93) = 2.25, p < 0.05). For cold water frequencies, ANOVA indicated a significant main effects of i.p. pregabalin pretreatment (F(3,31) = 13.4, p < 0.001) and time (F(3,93) = 8.70, p < 0.001, but a non-significant treatment X time interaction (F(9,93) = 1.83, p > 0.05). Post hoc comparisons revealed that compared to vehicle (n=13), 10 (n=7) and 100 (n=9) mg/kg pregabalin pretreatment on day 4 and 10 mg/kg on day 8 significantly reduced mechanical hypersensitivity (A) after CCI (*p < 0.05, n=6 for 30 mg/kg), and cold hypersensitivity (B) was significantly reduced by 10 mg/kg pregabalin on day 4, 8 and 12 after CCI (*p < 0.05) as compared to vehicle (n’s same as in A).

Fig. 3

Effect of intrathecal (i.t.) pregabalin post-treatment on mechanical (A) and cold (B) sensitivity in rats after chronic constriction injury (CCI) of sciatic nerve. For von Frey thresholds, ANOVA indicated a significant main effects of i.t. pregabalin post-treatment (F(3,48) = 2.97, p < 0.05) and time (F(4,192) = 36.1, p < 0.001, as well as a significant treatment X time interaction (F(12,192) = 2.19, p < 0.05). For cold water frequencies, ANOVA indicated a non-significant main effect of i.t. pregabalin post-treatment (F(3,47) = 0.61, p > 0.05), but a significant main effect of time (F(4,92) = 26.9, p < 0.001, and a significant treatment X time interaction (F(12,92) = 2.68, p < 0.01). Post hoc comparisons revealed that compared to vehicle (n=17) there was a significant reduction in mechanical hypersensitivity (A) with 10 (n=8) and 100 (n=18) μg i.t. pregabalin pretreatment on day 12, and 10 μg on day 16 after CCI (*p < 0.05, n=9 for 30 μg), and cold hypersensitivity (B) was significantly reduced by 100 μg pregabalin on day 12 after CCI (*p < 0.05) as compared to vehicle (n’s same as in a, except vehicle n=16).

Fig. 4

Effect of systemic (i.p.) pregabalin post-treatment on mechanical (A) and cold (B) sensitivity in rats after chronic constriction injury (CCI) of sciatic nerve. For von Frey thresholds, ANOVA indicated a significant main effects of i.p. pregabalin post-treatment (F(3,26) = 16.9, p < 0.001) and time (F(4,104) = 7.29, p < 0.001, as well as a significant treatment X time interaction (F(12,104) = 3.73, p < 0.001). For cold water frequencies, ANOVA indicated a non-significant main effects of i.p. pregabalin post-treatment (F(3,24) = 0.31, p > 0.05), but a significant main effect of time (F(4,96) = 20.8, p < 0.001, and a non-significant treatment X time interaction (F(12,96) = 1.61, p > 0.05). Post hoc comparisons revealed that compared to vehicle (n=13), 30 (n=6) and 100 (n=9) mg/kg pregabalin post-treatment (n=8) on days 8, 12 and 16 and 10 (n=7) mg/kg on day 16 significantly reduced mechanical hypersensitivity (A) after CCI (*p < 0.05), and cold hypersensitivity (B) was not significantly reduced by any pregabalin post-treatment on any day after CCI as compared to vehicle (n’s same as in a).

Fig. 5

Effect of systemic (i.p.) pregabalin pretreatment on behavioral nociceptive scores in the formalin test. Repeated measures ANOVA reveals a significant main effects of i.p. pregabalin dose (F(3,22) = 3.21, p < 0.05) and time (F(11,242) = 22.5, p < 0.001), but a non-significant dose X time interaction (F(33,242) = 1.27 p > 0.05). Post hoc comparison revealed that compared to vehicle (n=6), formalin test nociceptive scores were significantly reduced by 100 mg/kg pregabalin (n=8) at 5, and 20–35 min post-formalin, and by 30 mg/kg pregabalin (n=6) at only 20 min post-formalin, while 10 mg/kg (n=6) was without effect.

Fig. 6

Effect of systemic pregabalin pretreatment (100 mg/kg, i.p.) on the glutamate (A) and aspartate (B) concentration in samples from spinal cord dorsal horn of naïve rats given a hind paw injection of 5.0% formalin. The line graph shows post-pregabalin/vehicle treatment, and post-formalin injection time points for group of rats treated with pregabalin (n=10 for glutamate, n=9 for aspartate) or vehicle (n=12 for both glutamate & aspartate). Arrows indicate timing of i.p. pregabalin/vehicle and intraplantar (i.pl.) formalin injections at −10 and 0 min, respectively. ANOVA reveals significant main effects of drug (F(1,20) = 5.10, p < 0.05) and time (F(14,280) = 6.58, p < 0.001), and a significant drug–time interaction (F(14,280) = 1.91, p < 0.05) in the assessment for glutamate (A). ANOVA reveals a non-significant main effects of drug (F(1,19) = 0.04, p > 0.05), but a significant main effect of time (F(14,266) = 4.05, p < 0.001), and a non-significant drug–time interaction (F(14,266) = 0.65, p > 0.05) in the assessment for aspartate (A). Post hoc comparisons (Fisher’s) indicated that pregabalin reduced glutamate levels at the 5 and 20–40 min time points after formalin (A), and did not significantly affect aspartate levels at any point after formalin (B) (*p < 0.05, **p < 0.01).

Fig. 7

Effect of intrathecal pregabalin pretreatment (100 μg, i.t.) on the glutamate (A) and aspartate (B) concentration in samples from spinal cord dorsal horn of CCI rats given a hind paw injection of 1.0% formalin. The line graph shows post-pregabalin/vehicle treatment, and post-formalin injection time points for group of rats treated with pregabalin (n=9) or vehicle (n=11). Arrows indicate timing of i.p. pregabalin/vehicle and intraplantar (i.pl.) formalin injections at −10 and 0 min, respectively. ANOVA reveals significant main effects of drug (F(1,20) = 4.15, p < 0.05) and time (F(14,266) = 3.54, p < 0.001), and a non-significant drug–time interaction (F(14,266) = 1.13, p > 0.05) in the assessment for glutamate (A). ANOVA reveals a non-significant main effects of drug (F(1,25) = 1.54, p > 0.05), but a significant main effect of time (F(14,350) = 5.76, p < 0.001), and a non-significant drug–time interaction (F(14,350) = 1.44, p > 0.05) in the assessment for aspartate (A). Post hoc comparisons (Fisher’s) indicated that pregabalin reduced glutamate levels at the 10–25 and 35 min time points after formalin (A), and aspartate levels at the 25 and 50–55 min time points after formalin (B) (*p < 0.05, **p < 0.01).