Gonadal hormone-dependent nociceptor sensitization maintains nociplastic pain state in female mice

Abstract

Nociplastic pain conditions develop predominantly in women. We recently established a murine nociplastic pain model by applying postinjury thermal (40°C) stimulation to an injured (capsaicin-injected) area, triggering a transition to a nociplastic pain state manifesting as persistent mechanical hypersensitivity outside of the previously injured area. The nociplastic pain state was centrally maintained by spinal microglia in males but peripherally by ongoing afferent activity at the previously injured area in females. Here, we investigated whether gonadal hormones are critical for the development of this peripherally maintained nociplastic pain state in females. Although the transition to a nociplastic pain state still occurred in ovariectomized females, the pain state was maintained neither by ongoing afferent activity at the previously injured area nor by spinal microglia. Estradiol reconstitution a week before the injury plus postinjury stimulation, but not after the transition had already occurred, restored the development of peripherally maintained nociplastic mechanical hypersensitivity in ovariectomized females. G protein-coupled estrogen receptor antagonism during the transition phase mimicked ovariectomy in gonad-intact females, whereas the receptor antagonism after the transition gradually alleviated the nociplastic mechanical hypersensitivity. At the previously injured area, afferents responsive to allyl isothiocyanate (AITC), a TRPA1 agonist, contributed to the maintenance of nociplastic mechanical hypersensitivity in gonad-intact females. In ex vivo skin-nerve preparations, only AITC-responsive afferents from the nociplastic pain model in gonad-intact females showed ongoing activities greater than control. These results suggest that gonadal hormones are critical for peripherally maintained nociplastic pain state in females by sensitizing AITC-responsive afferents to be persistently active.

Figure 1:. Timeline for behavioral experiments examining female-specific mechanisms of nociplastic mechanical hypersensitivity

Two weeks before capsaicin plus 40°C postinjury stimulation (Cap+40°C, red arrow), female mice were ovariectomized (OVX, black arrow) or sham-ovariectomized (Sham-OVX, black arrow). A subset of OVX females were implanted with osmotic mini pumps containing 17β-estradiol (E2, purple arrows) either 7 days prior to Cap+40°C (day −7) or 4 days post-Cap+40°C (day 4, delayed E2). In another series of experiments, gonad-intact female mice received the estrogen receptor GPER antagonist G-36 (green arrows) either during (from day 0 to 3 post-capsaicin+40°C) or after (from day 4 to 6 post-Cap+40°C) the transition to a nociplastic pain state. The effect of various drugs on nociplastic mechanical hypersensitivity were determined days 7 to 10 post-Cap+40°C. Mechanical hypersensitivity was assessed using von Frey filaments (VFF, yellow arrow).

Figure 2:. Ovariectomy does not prevent the transition to a nociplastic pain state by postinjury stimulation

In both sham-ovariectomized (Sham-OVX, A) females and ovariectomized (OVX, B) females, postinjury thermal (40°C, yellow arrow) stimulation of a capsaicin (Cap, 0.1%, red arrow)-injected area made capsaicin-induced mechanical hypersensitivity persistent. **P<0.01, Cap control vs Cap+40°C by sequential Sidak test. Data are presented as median with interquartile range. Sham-OVX females: n = 7 per group. OVX females: n = 7 per group until 72 hours, after which n = 5 in Cap control and n = 4 in Cap+40°C.

Figure 3:. Ovariectomy makes nociplastic mechanical hypersensitivity independent of either ongoing peripheral afferent firing or spinal microglia

Local injection of bupivacaine (Bup, 0.75%, black arrow) at the previously capsaicin-injected area 7 to 10 days after capsaicin+40°C attenuated nociplastic mechanical hypersensitivity outside of the previously capsaicin-injected area in sham-ovariectomized (Sham-OVX, A) but not ovariectomized (OVX, B) females. Intrathecal injection of the microglia inhibitor Mac-1-saporin (Mac-1-Sap) effectively alleviated the nociplastic mechanical hypersensitivity in (C) males but not in (D) OVX females. ##P<0.01 vs pre-drug level by sequential Sidak test. Data are presented as median with interquartile range. Sham-OVX females: n = 5 per group. OVX females: n = 5 in vehicle (Veh), n = 6 in Bup, n = 3 in unconjugated saporin (Sap) and n = 4 in Mac-1-Sap. Males: n=4 per group.

Figure 4:. Estrogen reconstitution prior to, but not after, postinjury stimulation is sufficient to induce the peripherally maintained nociplastic pain state

In ovariectomized females supplemented with 17β-estradiol for one week prior to capsaicin+40°C (OVX+E2, A), nociplastic mechanical hypersensitivity outside of the previously capsaicin-injected area was attenuated by silencing peripheral afferents at the previously capsaicin-injected area with bupivacaine (Bup, 0.75%, black arrow) 7 to 10 days after capsaicin+40°C. Ovariectomized females were reconstituted with either 17β-estradiol or vehicle 4 days after capsaicin+40°C (OVX+delayed E2, B). When both 17β-estradiol reconstituted and non-reconstituted OVX females received Bup injection at the previously capsaicin-injected area (black arrow) 10 days after capsaicin+40°C, nociplastic mechanical hypersensitivity was not attenuated in 17β-estradiol reconstituted OVX females. ##P<0.01 vs pre-drug level by sequential Sidak test. Data are presented as median with interquartile range. OVX+E2 females, n = 5 per group. OVX+delayed E2, n = 5 in vehicle (Veh) and n = 4 in Bup. E2, 17β-estradiol.

Figure 5:. Estrogen receptor GPER activity is critical for both the development and maintenance of the peripherally maintained nociplastic pain state in gonad-intact females

(A) Antagonism of the estrogen receptor GPER with G-36 (50 μg/kg) during the transition phase (days 0–3 or 0, 1, 2, and 3 post-capsaicin+40°C) made gonad-intact females develop nociplastic mechanical hypersensitivity (outside of the previously injured area) that was not alleviated by bupivacaine injection (Bup, 0.75%, black arrow) at the previously capsaicin-injected area 7 to 10 days post-capsaicin. (B) After the transition to a nociplastic pain state, systemic injections of G-36 (green arrows) on days 4, 5, and 6 post-capsaicin+40°C gradually reduced the magnitude of nociplastic mechanical hypersensitivity in gonad-intact females. ##P<0.01 vs pre-drug level, **P<0.01 vs vehicle (Veh) by sequential Sidak test. Data are presented as median with interquartile range. Early G-36: n = 4 in Veh and n = 5 in G-36. Delayed G-36: n = 5 per group.

Figure 6:. Silencing AITC-responsive afferents alleviates nociplastic mechanical hypersensitivity in gonad-intact females 7 to 10 days post-capsaicin+40°C

Co-injection of QX-314 (2%, black arrow) with (A) allyl isothiocyanate (AITC, 30 μM), but not (B) capsaicin (Cap, 0.1%) or (C) flagellin (0.9 μg), at the previously capsaicin-injected area 7 to 10 days post-capsaicin+40°C significantly alleviated nociplastic mechanical hypersensitivity outside of the previously capsaicin-injected area in gonad-intact females. ##P ≤0.01 vs. pre-drug level by sequential Sidak test. Data are presented as median with interquartile range. Vehicle (Veh, QX-314), QX-314 + AITC, and QX-314 + cap, n = 6. QX-314 + flagellin, n = 5.

Figure 7:. AITC-responsive afferents innervating the previously injured area show increased ongoing activity 7 to 10 days post-capsaicin+40°C

Representative ongoing firing traces of AITC-responsive afferents in control (Con, A) and capsaicin+40°C group (Cap+40, B) from gonad-intact females. The spike shape templates are shown in the insets. (C) There was a significant increase in the mean ongoing firing frequency of AITC-responsive units in Cap+40 group, as compared to the control group and capsaicin alone group (Cap). This increase in ongoing firing frequency was not observed in OVX females. (D) There were no significant differences between groups in the mean ongoing firing frequency of AITC-nonresponsive units in either gonad-intact or OVX females. *P<0.05 vs. gonad-intact female Con. Data are presented as individual values with mean ± SEM. AITC-responsive units: for gonad-intact females: 16 units from n = 7 in Con, 18 units from n = 6 in Cap, 22 units from n = 8 in Cap+40; for OVX females: 9 units from n = 6 in Con, 15 units from n = 8 in Cap, and 21 units from n = 10 in Cap+40. AITC-nonresponsive units: for gonad-intact females: 29 units from n = 11 in Con, 18 units from n = 11 in Cap, 11 units from n = 8 in Cap+40; for OVX females 26 units from n = 9 in Con, 26 units from n = 8 in Cap, and 30 units from n = 10 in Cap+40.

Figure 8:. The mechanosensitive properties of AITC-responsive afferents innervating the previously injured area is not altered 7 to 10 days post-capsaicin+40°C

(A) Representative trace of ramp mechanical stimulation-evoked action potential firing from an AITC-responsive unit. The spike shape template is shown in the inset. There were no differences in the mechanical threshold between groups in either (B) AITC-responsive or (C) AITC-nonresponsive population. There were no significant differences in the number of ramp mechanical stimulation-evoked action potentials between groups in (D) AITC-responsive or (E) AITC-nonresponsive population. Data are presented as individual values with mean ± SEM. AITC-responsive units: for gonad-intact females: 25 units from n = 8 in control (Con), 27 units from n = 7 in Cap, 21 units from n = 10 in Cap+40; for OVX females: 18 from units from n = 6 in Con, 16 units from n = 7 in Cap, and 22 units from n = 10 in Cap+40. AITC-nonresponsive units: for gonad-intact females: 39 units from n = 15 in Con, 27 units from n = 12 in Cap, 21 units from n = 8 in Cap+40; for OVX females 35 units from n = 10 in Con, 38 units from n = 10 in Cap, and 46 units from n = 11 in Cap+40.