Acetazolamide and midazolam act synergistically to inhibit neuropathic pain

Abstract

Treatment of neuropathic pain is a major clinical challenge that has been met with minimal success. After peripheral nerve injury, a decrease in the expression of the K-Cl cotransporter KCC2, a major neuronal Cl(-) extruder, leads to pathologic alterations in GABA(A) and glycine receptor function in the spinal cord. The down-regulation of KCC2 is expected to cause a reduction in Cl(-) extrusion capacity in dorsal horn neurons, which, together with the depolarizing efflux of HCO(3)(-) anions via GABA(A) channels, would result in a decrease in the efficacy of GABA(A)-mediated inhibition. Carbonic anhydrases (CA) facilitate intracellular HCO(3)(-) generation and hence, we hypothesized that inhibition of CAs would enhance the efficacy of GABAergic inhibition in the context of neuropathic pain. Despite the decrease in KCC2 expression, spinal administration of benzodiazepines has been shown to be anti-allodynic in neuropathic conditions. Thus, we also hypothesized that spinal inhibition of CAs might enhance the anti-allodynic effects of spinally administered benzodiazepines. Here, we show that inhibition of spinal CA activity with acetazolamide (ACT) reduces neuropathic allodynia. Moreover, we demonstrate that spinal co-administration of ACT and midazolam (MZL) act synergistically to reduce neuropathic allodynia after peripheral nerve injury. These findings indicate that the combined use of CA inhibitors and benzodiazepines may be effective in the clinical management of neuropathic pain in humans.

Fig. 1

Intrathecal acetazolamide (ACT) inhibits neuropathic allodynia in the spinal nerve ligation (SNL) model. (A) Predrug mechanical withdrawal thresholds were measured and ACT was injected intrathecally and mechanical withdrawal thresholds were reassessed at the indicated time points. A significant main effect of ACT was observed (F(4, 144) = 6.95, p < 0.0001). (B) ACT, at the highest dose given, did not alter mechanical withdrawal thresholds in sham animals. (C) ACT dose-dependently inhibited neuropathic allodynia with an EC₅₀ (D) of 1.85 ± 3.3 μg. ***p < 0.001 with one-way ANOVA and Dunnett’s post hoc test.

Fig. 2

Acetazolamide (ACT), midazolam (MZL) and ACT and MZL in combination do not impair motor performance. The rotarod test was used to assess whether maximum effective doses of ACT or MZL impaired motor performance. Rats with intrathecal catheters were trained as described in the text and VEH, ACT, MZL or ACT + MZL (combined EC₅₀) were administered and rotarod performance was assessed at the indicated time points. Neither ACT, MZL nor ACT (0.97 ng) + MZL (1.85 μg) impaired motor performance as compared to vehicle injection.

Fig. 3

Intrathecal midazolam (MZL) inhibits neuropathic allodynia in the spinal nerve ligation (SNL) model. (A) Predrug mechanical withdrawal thresholds were measured and MZL was injected intrathecally and mechanical withdrawal thresholds were reassessed at the indicated time points. A significant main effect of MZL was observed (F(5, 246) = 6.31, p < 0.0001). (B) MZL, at the peak effect dose, did not alter mechanical withdrawal thresholds in sham animals. (C) MZL dose-dependently inhibited neuropathic allodynia with an EC₅₀ (D) of 0.97 ± 0.72 ng. *p < 0.05, **p < 0.01 with one-way ANOVA and Dunnett’s post hoc test.

Fig. 4

Intrathecal acetazolamide (ACT) combined with midazolam (MZL) acts synergistically to inhibit neuropathic allodynia in the spinal nerve ligation (SNL) model. (A) Predrug mechanical withdrawal thresholds were measured and ACT + MZL, at a fixed dose ratio of 1850–0.97 ng (see text for explanation) was injected intrathecally and mechanical withdrawal thresholds were reassessed at the indicated time points. A significant main effect of ACT + MZL was observed (F(3, 126) = 6.29, p = 0.0005). (B) ACT + MZL dose-dependently inhibited neuropathic allodynia with a combined dose EC₅₀ (C) of 190 ± 141 ng, curves for ACT and MZL alone are shown for comparison. (D) Isobolographic analysis revealed that ACT and MZL act synergistically to inhibit neuropathic allodynia. The A₅₀ for ACT given alone was 2.19 ± 0.75 μg. The A₅₀ of MZL was 1.07 ± 0.88 ng. The theoretical additive A₅₀ for a combined dose was 1058 ± 480 ng while the observed A₅₀ for ACT and MZL administered together was 182 ± 65 ng. The theoretical additive mixture A₅₀ differed from the observed A₅₀ significantly (p < 0.001) indicating that ACT and MZL act synergistically to inhibit neuropathic allodynia. (E) MZL, ACT or MZL + ACT were given intrathecally and paw withdrawal thresholds were measured at 60 min post-drug administration to test whether ACT would restore efficacy of MZL at a dose where efficacy was lost. ACT alone and ACT + MZL significantly reversed neuropathic allodynia while MZL alone did not. *p < 0.05, **p < 0.01 with one-way ANOVA and Dunnett’s post hoc test.