Inhibition of multiple voltage-gated calcium channels may contribute to spinally mediated analgesia by epipregnanolone in a rat model of surgical paw incision

Abstract

Voltage-activated calcium channels play an important role in excitability of sensory nociceptive neurons in acute and chronic pain models. We have previously shown that low-voltage-activated calcium channels, or T-type channels (T-channels), increase excitability of sensory neurons after surgical incision in rats. We have also found that endogenous 5β-reduced neuroactive steroid epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one] blocked isolated T-currents in dorsal root ganglion (DRG) cells in vitro, and reduced nociceptive behavior in vivo, after local intraplantar application into the foot pads of heathy rats and mice. Here, we investigated if epipregnanolone exerts an antinociceptive effect after intrathecal (i.t.) application in healthy rats, as well as an antihyperalgesic effect in a postsurgical pain model. We also studied if this endogenous neurosteroid blocks currents originating from high voltage-activated (HVA) calcium channels in rat sensory neurons. In in vivo studies, we found that epipregnanolone alleviated thermal and mechanical nociception in healthy rats after i.t. administration without affecting their sensory-motor abilities. Furthermore, epipregnanolone effectively reduced mechanical hyperalgesia after i.t application in rats after surgery. In subsequent in vitro studies, we found that epipregnanolone blocked isolated HVA currents in nociceptive sensory neurons with an IC₅₀ of 3.3 μM in a G-protein-dependent fashion. We conclude that neurosteroids that have combined inhibitory effects on T-type and HVA calcium currents may be suitable for development of novel pain therapies during the perioperative period.

Keywords: Neurosteroids; calcium; hyperalgesia; low voltage-activated calcium channels.

Figure 1.

The chemical structure of epipregnanolone.

Figure 2.

Intrathecal application of epipregnanolone reduces thermal and mechanical nociception in healthy rats. (a). Schematic representation of experimental protocol. Baseline measurements of responses to heat and mechanical stimuli were measured over two days before injection. Following an intrathecal (i. t.) injection of epipregnanolone or vehicle, thermal and mechanical nociception were assessed over time. (b). Graph of paw withdrawal latency (PWL) following i.t. injection of vehicle or epipregnanolone at 3, 100, and 300 µM, each applied in a volume of 50 µl, showing epipregnanolone alleviates thermal nociception in a dose dependent manner (n = 9–12 animals per group; two-way RM ANOVA with Sidak’s post-hoc test; interaction: F(12,144) = 2.498, p = 0.0053; treatment: F(3,36) = 7.123, p < 0.001; at 30 min: vehicle vs 100 µM: *** p < 0.001, vehicle vs 300 µM: *** p < 0.0001; at 60 min: vehicle vs 100 µM: ** p = 0.0064; vehicle vs 300 µM: ** p = 0.0038). (c). Graph of paw withdrawal response (PWR) following i.t. injection of vehicle or 300 µM epipregnanolone, each applied in a volume of 50 µl, showing the antinociceptive effect of epipregnanolone on mechanical nociception over time (n = 5–7 animals per group; two-way RM-ANOVA with Sidak’s post-hoc test; interaction: F(3,30) = 3.491; p = 0.0277; treatment: F(1,10) = 43.43, p < 0.0001; at 30 min: vehicle vs 300 µM: ** p = 0.0054; at 60 min: vehicle vs 300 µM: *** p < 0.001; at 90 min: vehicle vs 300 µM:*** p < 0.001). Each data point represents the mean ± SEM.

Figure 3.

Intrathecal application of epipregnanolone does not impair sensory-motor abilities of rats. (a). Schematic representation of experimental protocol for baseline measures of sensorimotor abilities prior to injection, and assessment performed after an intrathecal injection (i.t.) of 50 µl of 300 µM epipregnanolone. (b). Bar graph demonstrates time rats remained on the plank before and after an i.t. injection of 50 µl of 300 µM epipregnanolone (n = 6 rats for each group). (c). Bar graph of walking initiation demonstrates time required for rats to place all four paws outside the labeled square before and after i.t. injection of 50 µl of 300 µM epipregnanolone (n = 6 rats for each group, paired t-test, p = 0.21). (d). Bar graph demonstrates time rats remained on elevated platform before and after i.t. injection of 50 µl of 300 µM epipregnanolone (n = 6 rats for each group) (e). Bar graph demonstrates time rats remained on inclined screen before and after i.t. injection of 50 µl of 300 µM epipregnanolone (n = 6 rats for each group).

Figure 4.

Single intrathecal application of epipregnanolone reduces mechanical nociception in incised rats. (a). Schematic representation of protocol. Baseline measurements of paw withdrawal response (PWR) following mechanical stimulus determined two days before the incision, and assessment post-injection in surgically incised rats. Intrathecal (i.t.) injections of vehicle or 300µM epipregnanolone, each applied in a volume of 50 µl, were performed 24 h post-incision followed by assessment of mechanical hyperalgesia at 30, 60, 90 and 120 minutes post-injection. (b). Graph of time-course for PWR following i.t. injection of vehicle or 300µM epipregnanolone, each applied in a volume of 50 µl, showing antihyperalgesic effect of epipregnanolone on mechanical nociception (n = 4–7 rats per group, two-way RM ANOVA, interaction: p = 0.61; treatment: F(1,9) = 15.01, ** p = 0.0038). Each data point represents the mean ± SEM.

Figure 5.

Epipregnanolone inhibits high-voltage-activated (HVA) calcium currents in rat sensory neurons in a G-protein-dependent fashion. (a). Representative traces of evoked HVA currents (Vh −50 mV, Vt −10 mV) recorded from dissociated DRG neurons of healthy animals; the traces were recorded before (control), 4 minutes after the application of 10 µM epipregnanolone, and after a 4 minute washout of the neurosteroid (wash). (b). The concentration-response curve shows the percentage of current inhibition achieved after application of a range of different concentrations of epipregnanolone. Each point is the average of at least three different cells (total n = 34 cells, the exact number of cells per group is presented on the graph) ± SEM; solid line indicates best fit using unrestricted concentration-response equation, giving 85% for maximal current inhibition (V_max), IC₅₀ of 3.3 µM, and Hill coefficient 1.5. (c). HVA current inhibition of dissociated DRG. Red bar indicates inhibition with 10 μM epipregnanolone in the presence of GTP in the internal solution (red bar) is approximately 70% (n = 11 cells). Inhibition of HVA currents by epipregnanolone is reduced in the presence of GTP-γ-S (white bar, n = 10 cells, p < 0.001, Unpaired t-test) or GTP-β-S (gray bar, n = 10 cells, p < 0.001, Mann-Whitney test), as compared to the red bar.