Inflammation-induced shift in the valence of spinal GABA-A receptor-mediated modulation of nociception in the adult rat

Abstract

The objective of this study was to assess the impact of persistent inflammation on spinal gamma-aminobutyric acid-A (GABA-A) receptor-mediated modulation of evoked nociceptive behavior in the adult rat. Nocifensive threshold was assessed with von Frey filaments applied to the dorsal surface of the hind paw. The GABA-A receptor agonist muscimol, the antagonist gabazine, the benzodiazepine receptor agonist midazolam, and antagonists PK11195 and flumazenil were administered spinally in the presence and absence of complete Freund's adjuvant (CFA)-induced inflammation. In naive rats, muscimol increased and gabazine decreased nociceptive threshold. After CFA, the effects of these compounds were reversed: Low doses of muscimol exacerbated the inflammation-induced decrease in nociceptive threshold and gabazine increased nociceptive threshold. Midazolam increased nociceptive threshold both in the presence and absence of inflammation. Flumazenil but not PK11195 blocked the analgesic effects of midazolam. These findings indicate that inflammation-induced changes in GABA-A signaling are complex and are likely to involve several distinct mechanisms. Rectifying the changes in GABA-A signaling may provide effective relief from hypersensitivity observed in the presence of inflammation.

Perspective: An inflammation-induced shift in spinal GABA-A receptor signaling from inhibition to excitation appears to underlie inflammatory pain and hypersensitivity. Use of GABA-A receptor selective general anesthetics in association with therapeutic interventions may be contraindicated. More importantly, rectifying the changes in GABA-A signaling may provide effective relief from inflammatory hypersensitivity.

Figure 1

Stimulus–response functions obtained from naïve rats (open symbols) and from rats (closed symbols) in which inflammation of the hindpaw had been induced with an injection of CFA, 3 days before nociceptive testing: Data are mean ± SEM. The mean fitted EF50 from naïve and CFA rats is plotted in larger symbols. The mean EF50 from naïve and CFA rats after spinal saline injection (squares) is also plotted for comparison. Each rat was stimulated with an ascending series of von Frey filaments and the data from rats in each group were pooled. Note that inflammation was associated with a leftward shift in stimulus response function while spinal administration of saline had little effect in either CFA inflamed or naïve rats. The number of rats studied in each group was 129, 123, 18 and 15 for Baseline Naïve, Baseline CFA, Saline Naïve and Saline CFA, respectively.

Figure 2

A. Spinal injection of the GABA-A receptor agonist, muscimol in naïve rats resulted in a dose dependent increase in EF50 which was significant at the lowest dose tested (ANOVA, p<0.01). Baseline data (B) for the group of naïve rats treated with muscimol, collected prior to the injection of muscimol are plotted for comparison. B. 72 hrs after the induction of inflammation with CFA, spinal muscimol resulted in a “u-shaped” dose response curve reflecting a significant decrease in EF50 at 0.1 µg and an increase in EF50 at 1 µg. Baseline data (B) for the group of inflamed rats treated with muscimol, collected prior to the injection of muscimol are plotted for comparison. The number of rats pooled for each data point is indicated in parentheses. Error bars are smaller than the symbols for several data points. ** is p < 0.01, Holm-Sidak post-hoc test.

Figure 3

A. Spinal injection of the GABA-A receptor antagonist, gabazine, in naïve rats resulted in a bi-modal dose-response curve which was associated with an elevation in the EF50 at low doses and a decrease in EF50 at higher doses. Baseline data (B) for the group of naïve rats treated with gabazine, collected prior to the injection of muscimol are plotted for comparison. B. Spinal injection of gabazine 72 hrs after CFA-induced inflammation resulted in a significant increase in EF50 (ANOVA; p<0.01). Baseline data (B) for the group of inflamed rats treated with gabazine, collected prior to the injection of muscimol are plotted for comparison. Note, data are plotted on different Y-axis scales in order to more clearly illustrate changes in EF50. The number of rats pooled for each data point is indicated in parentheses. Error bars are smaller than the symbols for several data points. ** is p < 0.01, Holm-Sidak post-hoc test.

Figure 4

A. Spinal injection of the benzodiazepine agonist, midazolam, in naïve rats resulted in a dose-dependent increase in EF50, which was significant at 6 ng, the lowest concentration tested (ANOVA; p<0.05). Baseline data (B) for the group of naïve rats treated with midazolam, collected prior to the injection of muscimol are plotted for comparison. B. Spinal injection of midazolam 72 hrs after CFA-induced inflammation resulted also in a significant increase in EF50 (ANOVA; p<0.5). Baseline data (B) for the group of inflamed rats treated with midazolam, collected prior to the injection of muscimol are plotted for comparison. Data are plotted on different Y-axis scales in order to more clearly illustrate changes in EF50. The number of rats pooled for each data point is indicated in parentheses. Error bars are smaller than the symbol for one data point. ** is p < 0.01, Holm-Sidak post-hoc test.

Figure 5

A. Spinal injections of peripheral benzodiazepine receptor antagonist, PK11195 (PK where PK Lo is 1.1 µg (n = 4) and PK Hi is 7.1 µg (n = 4)), in naïve rats did not produce a shift in the EF50 relative to saline (sal) injected rats (n = 18). The combination of either 1.1 µg (n = 5) or 7.1 µg (n = 4) PK11195 with 20 ng midazolam (MDZ) resulted in an increase in EF50 that was not significantly different than that induced by 20 ng midazolam alone (17). B. 72 hours after CFA injection neither 1.1 µg (PK Lo, n = 3) nor 7.1 µg (PK Hi, n = 3) attenuated the increase in nociceptive threshold induced by 20 ng midazolam. Rather, this combination resulted in a further increase in nociceptive threshold that was significantly larger at the 1.1 µg dose than midazolam alone (n = 17). When given alone, neither the 1.1 µg dose (n = 4) nor the 7.1 µg dose (n = 3) had effects different from saline (n = 21). The start and end of each horizontal bar indicates groups that are significantly different based on post-hoc (Holm-Sidak) comparisons. ** is p < 0.01.

Figure 6

A. In naïve rats, spinal injection of the central benzodiazepine receptor antagonist, flumazenil (12 ng, Flu, n = 6) alone, or in combination with midazolam (20 ng, n = 6) had an influence on EF50 that was not significantly different from that produced by saline (Sal, n = 18). The increase in EF50 induced by midazolam alone (20 ng, n = 17) was significantly larger than that induced by saline or flumazenil alone. D. 72 hours following CFA injection, spinal injection of flumazenil (12 ng) with midazolam (Flu/MDZ, n = 6) significantly attenuated the increase in EF50 associated with midazolam (20 ng) alone (n = 17). Flumazenil (12 ng) alone (n = 7) had an effect on EF50 that was not significantly different from that produced by saline (n = 18). The start and end of each horizontal bar indicates groups that are significantly different based on post-hoc (Holm-Sidak) comparisons. * = p < 0.05, ** = p < 0.01.