Oxytocin Relieves Neuropathic Pain Through GABA Release and Presynaptic TRPV1 Inhibition in Spinal Cord

Abstract

Objective: Oxytocin (OT) is synthesized within the paraventricular nucleus and supraoptic nucleus of the hypothalamus. In addition to its role in uterine contraction, OT plays an important antinociceptive role; however, the underlying molecular mechanisms of antinociceptive role of OT remain elusive. We hypothesized that the antinociceptive effect of OT on neuropathic pain may occur via inhibition of TRPV1 activation in the spinal cord. The present study explores the antinociceptive role of OT and its mechanisms in neuropathic pain. Methods: Partial sciatic nerve ligation (pSNL) was performed to induce neuropathic pain. Animal behaviors were measured using a set of electronic von Frey apparatus and hot plate. Electrophysiological recordings and molecular biological experiments were performed. Results: Intrathecal administration of OT alleviated both mechanical allodynia and thermal hyperalgesia in pSNL rats (n = 6, per group, P < 0.0001, saline vs. OT group). Electrophysiological data revealed that OT significantly inhibited the enhancement of frequency and amplitude of spontaneous excitatory post-synaptic currents induced presynaptically by TRPV1 activation in the spinal cord. Moreover, the inhibitory effect of OT on capsaicin-induced facilitation of excitatory transmission was blocked by co-treatment with saclofen, while intrathecal administration of OT dramatically inhibited capsaicin-induced ongoing pain in rats, (n = 6, per group, P < 0.0001, saline vs. OT group). The paw withdrawal latency in response to heat stimulation was significantly impaired in TRPV1KO mice 3 days after pSNL upon OT (i.t.) treatment, compared with wild type mice (n = 6, P < 0.05). Finally, OT prevented TRPV1 up-regulation in spinal cords of pSNL model rats. Conclusion: OT relieves neuropathic pain through GABA release and presynaptic TRPV1 inhibition in the spinal cord. OT and its receptor system might be an intriguing target for the treatment and prevention of neuropathic pain.

Keywords: GABA; TRPV1; mechanical allodynia; neuropathic pain; oxytocin; spinal cord; thermal hyperalgesia.

FIGURE 1

Behavioral tests and OT concentrations in CSF in rats after pSNL. (A,B) Mechanical (A) and thermal tests (B) in rats from sham and pSNL groups, n = 8. (C) OT concentrations in cerebrospinal fluid (CSF) of rats from sham and pSNL groups, n = 6. Mean ± SEM, ^∗P < 0.05, ^∗∗P < 0.01 vs. sham group at the same time point. Repeated measures ANOVA followed by Dunnett’s post hoc test were performed; statistical differences by Dunnett’s post hoc test were shown.

FIGURE 2

Effects of intrathecal injection of OT on rat behavioral tests. (A,B) Mechanical (A) and thermal tests (B) in naïve rats following one dose of OT administered intrathecally. (C,D) Mechanical tests (C) and thermal tests (D) in rats treated with or without intrathecal injection of OT at the third day on pSNL. (E,F) Mechanical tests (E) and thermal tests (F) in rats treated with or without continuous (twice per day for 3 days) intrathecal injection of OT. Mean ± SEM, n = 8; ^∗P < 0.05, ^∗∗P < 0.01 vs. pSNL group at the same time point. Repeated measures ANOVA followed by Dunnett’s post hoc test were performed; statistical differences by Dunnett’s post hoc test were shown.

FIGURE 3

Effect of OT on capsaicin-induced facilitation of spontaneous excitatory transmission in Lamina II neurons. (A) Effect of OT (1 μM) on glutamatergic spontaneous excitatory transmission in Lamina II neurons. Lower panel is the emphasized current traces as the dash line indicated. (B,C) Analyzed data of the frequency (B) and amplitude (C) of spontaneous EPSC after OT application in Lamina II neurons. Mean ± SEM, n = 6. Paired Student’s t-test. (D) Effect of capsaicin (1 μM) on glutamatergic spontaneous excitatory transmission in Lamina II neurons. Lower panel is the emphasized current traces as the dash line indicated. (E) Effect of OT (1 μM) on capsaicin-induced glutamatergic spontaneous excitatory transmission in Lamina II neurons. Lower panel is the emphasized current traces as the dash line indicated. (F,G) Analysis of the frequency (F) and amplitude (G) of spontaneous EPSC after OT co-application with or without capsaicin in Lamina II neurons. Mean ± SEM, n = 11; ^∗P < 0.05, ^∗∗P < 0.01. One-way ANOVA followed by two-tailed t-test with Bonferroni correction.

FIGURE 4

Electrophysiological recording in Lamina II neurons of rat spinal cord upon different pharmacological treatments. (A) Effect of OT (1 μM) on GABAergic spontaneous transmission in the presence of strychnine (1 μM), a glycine-receptor antagonist. (B) Effect of OT (1 μM) on mininature GABAergic transmission in the presence of strychnine (1 μM) and TTX (0.5 μM). (C,D) Analyzed data of the frequency (C) and amplitude (D) of spontaneous and mininature IPSC before and after OT application in Lamina II neurons. Mean ± SEM, n = 8. One-way ANOVA followed by two-tailed t-test with Bonferroni correction. (E) Effect of OT (1 μM) on capsaicin-induced glutamatergic spontaneous excitatory transmission in Lamina II neurons. (F) Effect of OT (1 μM) on capsaicin-induced mininature glutamatergic excitatory transmission in Lamina II neurons. (G,H) Analyzed data of the frequency (G) and amplitude (H) of capsaicin-induced spontaneous and mininature EPSC before and after OT application in Lamina II neurons. Mean ± SEM, n = 6. One-way ANOVA followed by two-tailed t-test with Bonferroni correction. (I) Effect of saclofen (50 μM) on the inhibitory effects of OT on capsaicin-induced glutamatergic spontaneous excitatory transmission in Lamina II neurons. (J,K) Analyzed data of the frequency (G) and amplitude (H) of capsaicin-induced spontaneous EPSC after saclofen co-application with or without OT in Lamina II neurons. Mean ± SEM, n = 6. One-way ANOVA followed by two-tailed t-test with Bonferroni correction.

FIGURE 5

Immunofluorescent staining of TRPV1 and GABA_B in spinal cord. (A–C) Representative images of immunofluorescent staining of TRPV1 (green, A); GABA_B receptor (red, B); and merged TRPV1 and GABA_B receptor (yellow, C) in spinal cord of naïve rat. (D–F) Magnified images of immunofluorescent staining of TRPV1 (green, D); and GABA_B receptor (red, E); and merged TRPV1 and GABA_B receptor (yellow, F) in spinal cord of naïve rat.

FIGURE 6

Effects of intrathecal injection of OT on capsaicin-induced licking behaviors in rats. (A) Time courses of capsaicin-induced licking time in rats treated with or without OT administered intrathecally. Mean ± SEM, n = 6; ^∗P < 0.05, ^∗∗P < 0.01 vs. saline group at the same time point. Repeated measures ANOVA followed by Dunnett’s post hoc test were performed; statistical differences by Dunnett’s post hoc test were shown. (B) Total licking time after capsaicin injection in rats treated with or without OT administered intrathecally. Mean ± SEM, n = 6; ^∗∗P < 0.01 vs. saline group. Unpaired Student’s t-test.

FIGURE 7

Effect of OT administration on the expression level of TRPV1 in rat spinal cords. (A) Representative images of immunohistochemistry staining of TRPV1 (red) in rat spinal cord after peripheral nerve injury treated intrathecally with saline or OT. (B) Western blot results of TRPV1 protein bands in rat spinal cord after peripheral nerve injury treated intrathecally with saline or OT. (C) Comparison of TRPV1 protein levels in rat spinal cord after peripheral nerve injury treated intrathecally with saline or OT. Mean ± SEM, n = 6; ^∗∗P < 0.01 vs. pre group; ^##P < 0.01 vs. pSNL group. One-way ANOVA followed by two-tailed t-test with Bonferroni correction.

FIGURE 8

Effects of intrathecal injection of OT on WT and TRPV1KO mice after peripheral nerve injury. (A) Relative response frequency to von Frey test (0.4 g) of wild-type (WT) and TRPV1 knock-out (TRPV1KO) mice on the third day after pSNL treated with or without OT. (B) Paw withdrawal latency to thermal stimuli of WT and TRPV1KO mice on the third day after pSNL treated with or without OT. BL, baseline. Mean ± SEM, n = 6; ^∗P < 0.05, ^∗∗P < 0.01. One-way ANOVA followed by two-tailed t-test with Bonferroni correction.