A prolactin-targeting antibody to prevent stress-induced peripheral nociceptor sensitization and female postoperative pain

Abstract

Scheduled surgeries elicit stress in many patients. Levels of preoperative stress, anxiety, and female gender are known risk factors for increased and prolonged postoperative pain. The mechanisms by which psychological stress increases postoperative pain, especially in women, remain unknown. We hypothesized that stress amplifies postoperative pain by sensitizing dorsal root ganglion (DRG) nociceptors. Prolactin (PRL) is a female-predominant neurohormone that is controlled by estrogen and stress. PRL signals at the prolactin receptor long (PRLR-L) and short (PRLR-S) isoforms to induce gene transcription and nociception, respectively. Critically, prolactin sensitizes female, but not male, murine, Macaque and human nociceptors, revealing an evolutionarily conserved mechanism with high translational potential for human therapy. Prior restraint stress (RS) increased the magnitude and duration of incisional injury-induced postoperative pain hypersensitivity in both male and female mice. In females, RS or incisional injury downregulated PRLR-L and increased PRL-dependent nociceptor excitability. Female selective inhibition of postoperative pain hypersensitivity was produced by a) pharmacological inhibition of pituitary PRL b) overexpression of DRG PRLR-L to bias PRL signaling away from PRLR-S and c) CRISPR/Cas9 editing of PRLR isoforms. PL200,019, our recently discovered monoclonal antibody against human PRL (hPRL), prevented hPRL-induced sensitization of human female nociceptors. Using female mice genetically modified to express hPRL, rather than murine PRL, PL200,019 prevented both stress and incisional injury-induced hypersensitivity. Preemptive inhibition of stress-induced nociceptor sensitization with a monoclonal antibody to sequester PRL can improve female postoperative pain, diminish the need for postoperative opioids and decrease the risks of transition to chronic pain.

Keywords: pain; prolactin; sensitization; sex differences; therapy.

Fig. 1.

Pharmacological inhibition of pituitary PRL release prevents dysregulation of PRLR isoforms and blocks mechanical hypersensitivity following incisional injury. (A) Western blot showing downregulation of PRLR-L in female mouse DRG 1 d after paw incision surgery. (B) Quantification of PRLR-L expression downregulation after paw incision. (C) Quantification showing no change in the expression of PRLR-S. (D) Timeline of cabergoline treatments and experimental manipulations pertaining to panels E–M. (E) Western blot showing expression of PRLR-L and PRLR-S in cabergoline treated female mouse DRG after paw incision. (F) Cabergoline treatment increases the expression of PRLR-L relative to vehicle-treated mice after paw incision. (G) There was no change in the expression of PRLR-S following treatment with cabergoline after paw incision. (H) Treatment with cabergoline blocks the emergence of postoperative pain hypersensitivity in female mice. (I) Quantification of the area under the curve (AUC) for female mice after incisional injury. (J) Spontaneous nocifensive behaviors after injection of capsaicin into the contralateral paw relative to the incisional injury. (K) Treatment with cabergoline had no effect on postoperative pain hypersensitivity in male mice following incisional injury. (L) Quantification of the AUC for male mice after incisional injury. (M) Evaluation of spontaneous nocifensive behaviors in male mice after injection of capsaicin into the contralateral uninjured paw. Data are displayed as mean ± SEM. Each data point represents an independent animal. N = 6–8 mice per group for biochemistry. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (H and K). Other comparisons used the Mann–Whitney test. *P < 0.05 and **P < 0.01. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 2.

Overexpression of PRLR-L protects from incisional injury–induced hypersensitivity in female mice. (A) Timeline of experimental manipulations including intrathecal injection of PRLR-L-GFP plasmid to overexpress the receptor. (B) Western blot showing increased expression of PRLR-L protein in the DRG of female mice after intrathecal injection of a plasmid encoding the receptor. (C) Intrathecal injection of PRLR-L plasmid in female mice 1 d before incisional injury followed by mechanical hypersensitivity testing. (D) Quantification of the AUC for the female mice in panel C. (E) Male mice were given an intrathecal injection of a plasmid encoding PRLR-L 1 d before incisional injury followed by mechanical hypersensitivity testing. (F) Quantification of the AUC for the animals shown in panel E. Data are shown as mean ± SEM. The number of animals used is indicated in the figure. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (C and E). All other comparisons used the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 3.

Restraint stress causes mechanical hypersensitivity in male and female mice and prolongs recovery from incisional injury–induced pain. (A) Experimental timeline. (B) Restraint stress (RS) produces transient hindpaw allodynia in female mice that returns to baseline by approximately day 7. (C) Prior exposure to restraint stress prolongs the recovery and enhances incisional injury–induced pain hypersensitivity in female mice. (D) Quantification of the AUC for female mice after incisional injury. (E) RS produces transient hindpaw allodynia in male mice that resolves by approximately day 7. (F) Prior exposure to stress also prolongs the recovery from incisional injury and increases the magnitude of incisional injury–related pain hypersensitivity. (G) Quantification of the AUC for male mice after incisional injury. Data are mean ± SEM. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (B, C, E, and F). All other comparisons used the Mann–Whitney test. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 4.

Downregulation of PRLR-L following stress and injury triggers enhanced responses to a low concentration of PRL selectively in female mouse DRG neurons. (A) Representative traces from small diameter female mouse DRG neurons during current clamp recordings while injecting 600 pA of current. These recordings are from the same mice used for behavioral experiments in figure 3. (B) Plot showing that DRG neurons of mice previously exposed to stress had increased excitability to a low dose of PRL. (C) Rheobase, the minimum current to fire a single action potential, was reduced in female mice from the group exposed to stress and treated with PRL in vitro. (D) The resting membrane potential (RMP) was not different between the treatment groups. (E) Representative current clamp traces from male mouse DRG neurons treated with low doses of PRL evoked at 600 pA. (F) Excitability plot showing that increasing numbers of action potentials are fired per current step but that low concentration PRL treatment does not affect male sensory neurons. (G) The rheobase was not affected in male sensory neurons treated with PRL. (H) The resting membrane potential was slightly more negative in male mouse DRG neurons that had previously received stress exposure. Data are mean ± SEM. (Scale bars are 20 mV and 200 ms.) The number of cells recorded is indicated in the figure. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (B and F). All other comparisons used the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 5.

Treatment with cabergoline to upregulate PRLR-L protects from stress priming and injury-induced pain hypersensitivity in female mice. (A) Experimental timeline showing when RS was performed, when cabergoline was administered, and when DRG were harvested for further analysis. (B) Evaluation of hindpaw allodynia in female mice treated either with vehicle or with cabergoline. (C) Treatment with cabergoline blocks the development of incisional injury–induced pain hypersensitivity compared to vehicle treated controls. (D) Quantification of the AUC for female mice after incisional injury. (E) Evaluation of hindpaw allodynia in male mice treated with cabergoline or vehicle after restraint stress. (F) Treatment with cabergoline does not affect the development of incisional injury–related pain hypersensitivity. (G) Quantification of the AUC for male mice after incisional injury. (H) Western blot showing the relative expression of PRLR-L and PRLR-S after treatment with vehicle or cabergoline (CB) in sham- or RS-treated female mice in the absence of incisional injury. (I) Quantification of the expression of PRLR-L showing that RS decreases expression of PRLR-L, treatment with cabergoline upregulates PRLR-L and that RS-induced reduction can be blocked by cabergoline treatment. (J) There were no changes in the level of PRLR-S expression with RS or cabergoline treatment. Data are mean ± SEM. The number of animals used for behavioral experiments is indicated in the figure. N = 8–9 mice for biochemistry. One-way ANOVA with Sidak’s test for multiple comparisons (I and J). Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (B, C, E, and F). All other comparisons used the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 6.

Treatment with cabergoline prevents hyperexcitability following overnight incubation with a low concentration of PRL. (A) Representative traces evoked at 600 pA from female mouse DRG neurons following treatment with low concentration PRL. These neurons were isolated from the same animals in which behavior was performed in Fig. 5. (B) Treatment with cabergoline reduced the excitability of female mouse DRG neurons in response to low concentration PRL compared to mice treated with vehicle. (C) Rheobase was not affected following treatment with low concentration PRL in cabergoline treated animals. (D) The resting membrane potential was slightly more negative in DRG neurons from female mice treated with PRL. (E) The action potential count at maximum current injection was significantly lower in female mice following treatment with cabergoline. (F) Representative traces evoked at 600 pA from male DRG neurons treated with low concentration PRL. (G) The excitability of male DRG neurons was not affected by PRL treatment in mice treated with either vehicle or cabergoline. (H) The rheobase and the (I) RMP of male mouse DRG neurons was not affected by PRL treatment. (J) The maximum number of action potentials fired was not significantly different between groups. Data are mean ± SEM. (Scale bars are 20 mV and 200 ms.) CBG, Cabergoline. The number of cells recorded is indicated in the figure. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (B and G). All other comparisons used the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 7.

Treatment with PRLR-total CRISPR elicits female-selective prevention of incisional injury–related pain hypersensitivity and DRG hyperexcitability in response to low concentration PRL treatment. (A) Experimental timeline. (B) Female mice were given an intrathecal injection of a CRISPR construct that deletes the PRL receptor (Prlr-total CRISPR) and this treatment blocked the development of RS-induced hindpaw allodynia. (C) Treatment with Prlr-total CRISPR also prevented the incisional injury–induced postoperative pain hypersensitivity following stress priming in female mice. (D) The AUC quantifying the differences observed in panel B following incisional injury. (E) Representative traces from female mouse DRG sensory neurons recorded after overnight treatment with a low dose of PRL and obtained from mice treated with control CRISPR or Prlr-total CRISPR and subjected to RS and incisional injury. (F) The excitability of female sensory neurons from mice treated with Prlr-total CRISPR was significantly lower relative to control CRISPR mice. Inset shows the 200 pA current step. (G) The rheobase was increased in sensory neurons from mice treated with Prlr-total CRISPR. (H) There was no change in the resting membrane potential in sensory neurons from mice treated with Prlr-total CRISPR compared to those of control treated mice. Data are mean ± SEM. (Scale bars are 20 mV and 200 ms.) The number of cells recorded is indicated in the figure. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (B, C, and F). All other comparisons used the Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional statistical details can be found in SI Appendix, Table S2.

Fig. 8.

Sequestration of PRL prevents RS induced hindpaw allodynia, incisional injury–related pain hypersensitivity and in vitro sensitization in human female sensory neurons. (A) Representative action potential traces of human DRG neurons treated with hPRL (50 nM) overnight or hPRL with the neutralizing antibody (25 nM). (B) Excitability of human sensory neurons showing that treatment with the neutralizing antibody blocks hPRL induced hyperexcitability. Inset shows the individual data points for the action potentials evoked at the 1,500 pA current step. (C) The rheobase was significantly higher in the group treated with the neutralizing antibody. (D) The resting membrane potential was unaffected by treatment with the neutralizing antibody. Data are shown as mean ± SEM. (Scale bars are 20 mV and 200 ms.) (E) Diagram showing the humanized mouse that releases hPRL instead of mouse PRL. The Right hand side shows the neutralizing antibody built on a mouse IgG backbone with complementarity determining region that recognizes hPRL. (F) Experimental timeline for stress, antibody, and incisional injury treatments. (G) Treatment with the neutralizing antibody PL 200,019 (20 mg/kg s.q.) in female humanized mice blocked the development of RS-induced hindpaw allodynia. (H) These humanized mice also demonstrated reduced postoperative pain hypersensitivity following treatment with PL 200,019. (I) The AUC was lower in the humanized mice treated with the neutralizing antibody. Two-way repeated measures ANOVA with Sidak’s test for multiple comparisons (B, G, and H). All other comparisons used the Mann–Whitney test. Data are mean ± SEM. The number of animals or cells is indicated in the figure. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Additional statistical details can be found in SI Appendix, Table S2.