17β-Estradiol Attenuates Neuropathic Pain Caused by Spared Nerve Injury by Upregulating CIC-3 in the Dorsal Root Ganglion of Ovariectomized Rats

Abstract

17β-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17β-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17β-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17β-estradiol administration was associated with increased expression of ClC-3. 17β-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17β-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17β-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.

Keywords: 17β-estradiol; ClC-3; neuropathic pain; ovariectomy; spared nerve injury.

FIGURE 1

Experimental model and schedule of drug intervention. (A) Ovarian removal (OVX) and spared nerve injury (SNI) model protocol. Normal female rats underwent ovariectomy 2 weeks before SNI. Two weeks later, OVX rats underwent SNI and behavioral testing at different time points of SNI; the ipsilateral L_4–6 dorsal root ganglion was obtained as tissue sample after behavioral testing. (B) After the SNI model was established, rats were treated with 17β-estradiol for 7 days (from day 0 to day 6, 30 μg/kg/day) subcutaneously. On the 7th day of SNI, intrathecal Cltx or DMSO (1 μM/day, 20 μL) was administered for 4 days. The L_4–6 dorsal root ganglia of rats were collected on the 7th day and 10th day of SNI after behavioral testing. OVX, ovariectomy; SNI, spared nerve injury; E2, 17β-estradiol; Cltx, Chlorotoxin; DMSO, vehicle, dimethyl sulfoxide.

FIGURE 2

Effects of ovariectomy (OVX), spared nerve injury (SNI), and drug treatment on ClC-3 expression and behavior. (A) No change in the thermal threshold was observed after OVX (n = 8 per group). (B) There was no significant difference in duration of paw lifting in response to a cold stimulus (n = 9 per group). (C) Increased sensitivity to cold stimulation started from the 3rd day after SNI and lasted until the end of behavioral testing with slight recovery. Estrogen administration partially reversed this pain allergy in SNI rats until the day 21. Intrathecal injection of ClC-3 specific inhibitor, Cltx, resulted in cold hyperalgesia recovery. Vehicle solution had no effect (n = 6 per group). ^∗∗∗P < 0.01, OVX + SNI vs. OVX group; ^##P < 0.01, ^###P < 0.001, OVX + SNI + E2 vs. OVX + SNI group; ^&⁣&⁣&P < 0.001, OVX + SNI + E2 + Cltx vs. OVX + SNI + E2 group; ⁺⁺⁺P < 0.001, OVX + SNI + E2 + Cltx vs. OVX + SNI group. (D) Thermal pain did not produce significant differences among all five groups. OVX, ovariectomy; SNI, spared nerve injury; PWTL, paw withdrawal thermal latency; PWCL, paw withdrawal cold latency; s.c., subcutaneous; i.t., intrathecal injection. (E) Western blot images of ClC-3 protein expression show that there were no significant differences after OVX; n = 6 per group. (F) Serum estrogen decreased significantly after ovariectomy; n = 6 per group), ^∗∗∗P < 0.001, OVX vs. Pre-OVX.

FIGURE 3

ClC-3 expression, mainly in medium/small-sized and in large DRG neurons. (A) Immunofluorescence double labeling revealed that the ClC-3 protein was colocalized with IB4 (a marker of non-peptidergic C-type neurons), CGRP (a marker of peptidergic C-type neurons), and NF-200 (a marker of A-type neurons). Arrows refer to co-labeled neurons, scale bar = 50 μm. (B) The percentage of IB4, CGRP, and NF-200 positive (green) neurons relative to ClC-3 (red) positive cells. (C) Neuronal diameter size of IB4, CGRP, and NF-200. DRG, dorsal root ganglion.

FIGURE 4

ClC-3 expression was decreased in a time-dependent manner in the ipsilateral L_4–6 DRG neurons of SNI rats after OVX. (A) Immunofluorescent signal of ClC-3 (red) detected in the DRG neurons of SNI rats after OVX. A1: Negative control (PBS). A2: OVX. A3: OVX + SNI D3. A4: OVX + SNI D7. A5: OVX + SNI D14. A6: OVX + SNI D21. PBS, Phosphate buffered saline; D3, 3 days after SNI; D7, 7 days after SNI; D14, 14 days after SNI; D21, 21 days after SNI; scale bar = 50 μm. (B) Western blot analysis showed that the ClC-3 protein levels were altered in a time-dependent manner. A significant decrease was detected on day 7 after SNI; n = 6 per group, ^∗∗∗P < 0.001, compared to OVX group. (C) Quantification of ClC-3 positive neurons in ipsilateral L_4–6 DRGs of OVX and OVX + SNI rats; ^∗∗P < 0.01, ^∗∗∗P < 0.001, compared to the OVX group.

FIGURE 5

Cltx inhibited the 17β-estradiol mediated increase in ClC-3 protein expression but not in mRNA after intrathecal injection. (A) Subcutaneously injected 17β-estradiol (once per day for 7 consecutive days at SNI Day 0 to Day 6) reversed the downregulation of ClC-3 protein caused by SNI. Intrathecal injection of Cltx (once per day for 4 consecutive days starting at the 7th day of SNI after 17β-estradiol injection) reduced estrogen-mediated protein increased expression. Vehicle solution had no effect. n = 6 per group, ^∗∗∗P < 0.001, OVX + SNI vs. OVX group; ^###P < 0.001, OVX + SNI + E2 vs. OVX + SNI group; ^&⁣&⁣&P < 0.001, OVX + SNI + E2 + Cltx vs. OVX + SNI + E2 group; E2: 17β-estradiol; Cltx, Chlorotoxin; D0, the day of SNI operation; D6, 6 days after SNI. (B) ClC-3 mRNA level decreased after SNI and was rescued by 17β-estradiol; repeated daily intrathecal injection of Cltx (1 μM) did not suppress ClC-3 mRNA level. Vehicle solution had no significant effect; n = 6 per group. ^∗∗∗P < 0.001, OVX + SNI vs. OVX group; ^##P < 0.01, OVX + SNI + E2 vs. OVX + SNI group.

FIGURE 6

Intrathecal Cltx repressed ClC-3 protein expression and aroused cold hyperalgesia in OVX rats and aggravated hyperalgesia in OVX + SNI rats. (A) Repeated daily intrathecal injection of Cltx caused cold hyperalgesia in OVX rats from the injection day until the end of behavioral testing with slight recovery. Intrathecal injection of Cltx daily from SNI day 7 to day 10 aggravated hyperalgesia on day 10 of SNI (n = 6 per group). ^∗∗∗P < 0.01, OVX + Cltx vs. OVX + DMSO group; ^#P < 0.05, OVX + SNI + DMSO vs. OVX + SNI + Cltx group; DMSO: vehicle, dimethyl sulfoxide; PWCL, paw withdrawal cold latency; OVX, ovariectomized; SNI, spared nerve injury. (B) No change in the thermal threshold was observed (n = 6 per group). PWTL, paw withdrawal thermal latency. (C) Intrathecal injection of Cltx decreased ClC-3 protein expression both in OVX rats and OVX + SNI rats; n = 6 per group, ^∗∗∗P < 0.001, OVX + Cltx vs. OVX + DMSO group; ^###P < 0.001, OVX + SNI + DMSO vs. OVX + SNI + Cltx group.

FIGURE 7

17β-estradiol attenuated increased excitability of DRG neurons in spared nerve injury ovariectomized rats and was inhibited by Cltx. Current threshold (rheobase) was determined as the current required for activating the first action potential. (A–F) On the right, representative traces of action potentials (APs) evoked by current injections into DRG neurons from OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO, and OVX + SNI + E2 + Cltx groups; n = 6 per group; On the right, twice in the figure, the number of action potentials produced at the corresponding 2 × rheobase.

FIGURE 8

17β-estradiol reversed current threshold (rheobase) decrease and the number of action potentials produced at the corresponding 2 × rheobase increase and was blocked by Cltx. (A) Statistical analysis revealed the rheobase. (B) Size of neurons. (C) Membrane capacitance. (D) Resting potential. (E) Number of action potentials (APs) at 2 × rheobase in DRG neurons. n = 6 per group, ^∗∗∗P < 0.001, OVX + SNI vs. OVX group. ^###P < 0.001, OVX + SNI + E2 vs. OVX + SNI group; ^&⁣&P < 0.01, ^&&&P < 0.001 OVX + SNI + E2 + Cltx vs. OVX + SNI + E2 group.

FIGURE 9

Schematic of potential 17β-estradiol-mediated mechanisms of neuropathic pain regulation in ovariectomized female rats. 17β-estradiol may regulate the expression of ClC-3 mRNA through the 17β-estradiol receptor, including nuclear receptors α and β or membrane receptor GPER, via certain signaling pathways in the cell, thereby affecting the expression of ClC-3 mRNA and protein and relieving neuropathic pain. However, this effect may be blocked by Cltx, a specific blocker that affects the function of ClC-3 channel protein but does not affect the expression of mRNA. GPER, G protein-coupled 17β-estradiol receptor. ER-α and ER-β: nuclear 17β-estradiol receptors α and β.