Neuroendocrine Mechanisms Governing Sex Differences in Hyperalgesic Priming Involve Prolactin Receptor Sensory Neuron Signaling

Abstract

Many clinical and preclinical studies report higher prevalence and severity of chronic pain in females. We used hyperalgesic priming with interleukin 6 (IL-6) priming and PGE₂ as a second stimulus as a model for pain chronicity. Intraplantar IL-6 induced hypersensitivity was similar in magnitude and duration in both males and females, while both paw and intrathecal PGE₂ hypersensitivity was more persistent in females. This difference in PGE₂ response was dependent on both circulating estrogen and translation regulation signaling in the spinal cord. In males, the duration of hypersensitivity was regulated by testosterone. Since the prolactin receptor (Prlr) is regulated by reproductive hormones and is female-selectively activated in sensory neurons, we evaluated whether Prlr signaling contributes to hyperalgesic priming. Using ΔPRL, a competitive Prlr antagonist, and a mouse line with ablated Prlr in the Nav1.8 sensory neuronal population, we show that Prlr in sensory neurons is necessary for the development of hyperalgesic priming in female, but not male, mice. Overall, sex-specific mechanisms in the initiation and maintenance of chronic pain are regulated by the neuroendocrine system and, specifically, sensory neuronal Prlr signaling.SIGNIFICANCE STATEMENT Females are more likely to experience chronic pain than males, but the mechanisms that underlie this sex difference are not completely understood. Here, we demonstrate that the duration of mechanical hypersensitivity is dependent on circulating sex hormones in mice, where estrogen caused an extension of sensitivity and testosterone was responsible for a decrease in the duration of the hyperalgesic priming model of chronic pain. Additionally, we demonstrated that prolactin receptor expression in Nav1.8⁺ neurons was necessary for hyperalgesic priming in female, but not male, mice. Our work demonstrates a female-specific mechanism for the promotion of chronic pain involving the neuroendrocrine system and mediated by sensory neuronal prolactin receptor.

Keywords: estrogen; nociceptor; prolactin; sex dimorphism; testosterone; translation regulation.

Figure 1.

Persistence of hyperalgesic priming is greater in female mice. A, Schematic of the IL-6-induced hyperalgesic priming model. BL, Baseline measurements. Brown arrows indicate injection time points. Blue arrows indicate post-PGE₂ mechanical nociception measurement time points. B, Hyperalgesic priming model: IL-6 priming into paw and PGE₂ injection into paw of female and male C57BL/6 mice. C, Hyperalgesic priming model: IL-6 priming into paw and PGE₂ injection into SC of female and male C57BL/6 mice. D, The same model as in C in female and male Institute of Cancer Research (ICR) mice. Arrows indicate injection time points for IL-6/Veh and PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: C, IL-6 male compared with Veh-female: ****p < 0.0001; IL-6 female compared with Veh-female: ★★★★p < 0.0001; ★★★p < 0.001; all others: *p < 0.05; ****p < 0.0001. n = 5–8.

Figure 2.

Contribution of gonadal hormones profoundly influences hyperalgesic priming in female and male mice. A, Hyperalgesic priming model with spinal PGE₂ injection in WT female, OVX, and OVX+E (B) GdX, and (C) WT male, GdX, and GdX-T C57BL mice. B, The disparity in timelines between GDX animals and those animals that are naive or GdX-T. Arrows indicate injection time points for IL-6 and PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: A, ***p < 0.001; ****p < 0.0001, for OVX-E compared with female; ★★p < 0.01 for OVX compared with female; C, ***p < 0.001; ★★p < 0.01 for GdX-T compared with male; n = 5 or 6. ★★★★p < 0.0001.

Figure 3.

Spinal local translation only contributes to hyperalgesic priming in intact female mice. A, Hyperalgesic priming model with spinal PGE₂ injection in WT male, (B) WT female, and (C) OVX mice. 4EGI-1 (10 µg) or vehicle was administered spinally at 30 min before PGE₂ injection. Arrows indicate injection time points for IL-6 and 4EGI-1/PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: ****p < 0.0001. n = 5 or 6.

Figure 4.

Peripheral PRL only induces hyperalgesic priming in female mice. A, Schematic of the PRL-induced hyperalgesic priming model. BL, Baseline measurements after PRL-induced hypersensitivity is fully resolved. Brown arrows indicate injection time points. Blue arrows indicate post-PGE₂ treatment mechanical nociception measurement time points. B, C, Hyperalgesic priming model; PRL or vehicle priming into paw and PGE₂ injection into SC of male (B) or female (C) C57BL mice. Arrows indicate injection time points for PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: ***p < 0.001; ****p < 0.0001. n = 5–7.

Figure 5.

Evidence for translocation of Prlr mRNA to sensory neuronal peripheral and central terminals in female mice. A, Schematic of Prlr^fl/fl and Nav1.8^cre/-/Prlr^fl/fl (Prlr CKO) genes and corresponding transcribed mRNA from these genes. Location of Prlr-F and GFP-R1 (red arrows) and GFP-F and GFP-R2 (blue arrow). B, Representative panel for PCR of PGP9.5 mRNA (Uchl1 gene) from total RNA isolated from DRG and hindpaws (HP) of Prlr^fl/fl (Con) and Prlr CKO female mice. B′, Quantification of data shown in B for Prlr CKO female mice (**p < 0.01; n = 3). C, Representative panel for PCR of hybrid 2500 bp Prlr mRNA using Prlr-F (exon 4) and GFP-R1 primers (see A) for DRG and SC total RNA isolated from Prlr^fl/fl (Con) and Prlr CKO of female mice. C′, Quantification of data shown in C for Prlr CKO female mice (**p < 0.01; n = 3). D, Representative panel for PCR of GFP mRNA using GFP-F and GFP-R2 primers (see A) for DRG and SC total RNA isolated from Prlr^fl/fl (Con) and Prlr CKO of female mice. D′, Quantification of data shown in D for Prlr CKO female mice (*p < 0.05; n = 3). E, Representative panel for PCR of GFP mRNA using GFP-F and GFP-R2 primers (see A) for DRG and HP total RNA isolated from Prlr^fl/fl (Con) and Prlr CKO of female mice. E′, Quantification of data shown in E for Prlr CKO female mice (**p < 0.01; n = 3).

Figure 6.

Regulation of hyperalgesic priming by sensory neuronal Prlr selectively in female mice. A, B, Hyperalgesic priming model with peripheral IL-6/Veh and spinal PGE₂ in Prlr^fl/fl (Prlr-lox; control) and Nav1.8^cre/-/Prlr^fl/fl (Prlr CKO) in male (A) and female (B) mice. Arrows indicate injection time points for IL-6/Veh and PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: A, ****p < 0.001, for Prlr-lox compared with Veh-Prlr-lox; and ★★★★p < 0.0001; ★★★p < 0.001, for Prlr CKO compared with Veh-Prlr-lox. B, *p < 0.05; **p < 0.01; ***p < 0.001. n = 4–7.

Figure 7.

Regulation of the initiation and maintenance of hyperalgesic priming by peripheral and spinal Prlr in female mice. Hyperalgesic priming model in female mice with peripheral IL-6 and spinal PGE₂. A, Schematic of injection locations and timing. B, Vehicle (no ΔPRL), Prlr antagonist (ΔPRL; 5 µg) was coadministered with IL-6 in paw or SC. C, Schematic of injection locations and timing. D, Vehicle (no ΔPRL) was injected into paw. ΔPRL (5 µg) was given into the paw or SC 30 min before spinal PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: *p < 0.05; ****p < 0.0001, for ΔPRL intrathecally compared with no ΔPRL; and ★★★★p < 0.0001; ★p < 0.05, for ΔPRL intraplantarly compared with no ΔPRL; n = 5–7).

Figure 8.

Lack of effect of systemic bromocriptine on hyperalgesic priming in female and male mice. A, B, Hyperalgesic priming model with peripheral IL-6 and spinal PGE₂ in vehicle and bromocriptine (i.p.; BrCre) treatments of male (A) and female (B) mice. Arrows indicate injection time points for IL-6 and PGE₂. Repeated-measures ANOVA with Bonferroni post hoc test: ***p < 0.001; ****p < 0.0001. n = 5 or 6.