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Men and women acquaintance affliction differently. However, the mechanisms mediating this aberration are unclear. The abbreviate isoform of the prolactin (PRL) receptor (PRLR-S), but not the continued isoform (PRLR-L), has been apparent to adapt the affect of acoustic neurons in changeable rodents. Here, Chen et al. advised opioid-induced hyperalgesia (OIH) and appear that opioid administration, but not trauma-induced assumption injury, aggrandized PRL and decreased PRLR-L in changeable animals, announcement the activation of PRLR-S and the development of OIH. PRL inhibition prevented the accident of OIH in changeable animals. The after-effects advance that targeting PRL signaling ability be an able appraisal for preventing OIH in women.



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Pain is added accustomed in women for affidavit that abide unclear. We accept articular a apparatus of injury-free nociceptor sensitization and opioid-induced hyperalgesia (OIH) answer by prolactin (PRL) in females. PRL signals through mutually inhibitory continued (PRLR-L) and abbreviate (PRLR-S) receptor isoforms, and PRLR-S activation induces neuronal excitability. PRL and PRLR announcement were college in females. CRISPR-mediated alteration of PRLR-L answer nociceptor sensitization and allodynia in naïve, complete changeable mice that depended on circulating PRL. Opioids, but not trauma-induced assumption injury, decreased PRLR-L announcement OIH through activation of PRLR-S in changeable mice. Abatement of both PRLR-L and PRLR-S (total PRLR) prevented, admitting PRLR-L overexpression rescued accustomed OIH selectively in females. Inhibition of circulating PRL with cabergoline, a dopamine D2 agonist, up-regulated PRLR-L and prevented OIH alone in females. The PRLR-L isoform accordingly confers aegis adjoin PRL-promoted affliction in females. Limiting PRL/PRLR-S signaling pharmacologically or with gene therapies targeting the PRLR may be able for abbreviation affliction in a female-selective manner.



Prolactin (PRL) is an endocrine polypeptide hormone that is appear from the pituitary and extrapituitary sources and is now accustomed to accept abounding biological functions above its role in lactation and affectionate behavior (1). The above physiological inhibitor of circulating PRL is dopamine, appear from tuberoinfundibular dopamine (TIDA) neurons in the arcuate base of the hypothalamus (2, 3). Dopamine is transported to the antecedent pituitary area it inhibits PRL beard from lactotrophs via dopaminergic D2 receptors (4). Patients with prolactinoma associated with hyperprolactinemia ache from added accident of cephalalgia and are advised by blurred circulating PRL with dopamine agonists (5, 6). In rodents and humans, another splicing generates PRLR-L (long receptor) and PRLR-S (short receptor) isoforms of PRLR that accept identical extracellular domains but adapt in the breadth and arrangement of their cytoplasmic domains (7). PRLR-S is anticipation to adapt affect of changeable acoustic neurons by accentuation of colocalized ion channels, including the brief receptor abeyant vanilloid 1 (TRPV1) (8, 9). Studies in heterologous announcement systems accept appropriate a alternate anatomic inhibition amid PRLR-L and PRLR-S through intracellular mechanisms (8, 10–12), although this abstraction has never been activated in vivo. Women accept college circulating PRL than men and greater acuteness to beginning affliction (13). Nevertheless, women do not frequently alive in pain. We speculated that a accessible about-face in the antithesis of PRLR isoform announcement during dissection affliction altitude may affect nociceptor sensitization and affliction in females.



Although opioids are acclimated to amusement pain, paradoxically, these drugs accept additionally been associated with nociceptor sensitization and allodynia, frequently referred to as opioid-induced hyperalgesia (OIH) (14, 15). OIH has been appear afterwards the use of opioids in both biologic abusers and in patients with affliction (16). An additional, acclaimed ancillary aftereffect of opioids is the disruption of hormonal activity that includes brief hyperprolactinemia and galactorrhea, best generally empiric in females (17, 18). We accordingly bent whether the PRL/PRLR arrangement ability be disrupted by opioid administering in the absence of injury. We acclimated a archetypal of trauma-induced neuropathic affliction as a comparator. We begin that announcement of PRLR isoforms is sexually dimorphic. Announcement of PRLR-L and PRLR-S were abundant greater in changeable than in macho afterwards base ganglia (DRGs). In females, abbreviating PRLR-L sensitized nociceptors and elicited allodynia. In addition, appraisal with opioids, but not assumption injury, answer nociceptor sensitization and allodynia through down-regulation of PRLR-L. Conversely, abbreviating circulating PRL added the announcement of PRLR-L and provided careful furnishings adjoin nociceptor sensitization and allodynia. Overexpression of PRLR-L antipodal OIH selectively in females. Thus, our studies acknowledge a apparatus of nociceptor sensitization, pain, and OIH in females and advance approaches for sex-based affliction therapies.

Consistent with antecedent letters (19, 20), we detected college serum PRL in changeable mice compared with males (Fig. 1A). Appliance PRLR-LCre/− beyond with GFPflx/− mice to anticipate PRLR-L–positive neurons, we empiric that the boilerplate cardinal of these beef was college in changeable than in macho mice (Fig. 1B). Appliance Western blemish analysis, we begin that both PRLR-S and PRLR-L isoforms were empiric in DRGs from naïve changeable mice. However, actual low announcement of PRLR-S and PRLR-L were detected in DRGs from males. Neither PRLR isoform was empiric in the afterwards lumbar analgesic bond in either females or males (Fig. 1C). The after-effects appearance sexually dimorphic announcement of PRL, as able-bodied as of PRLR isoforms, and accommodate a base for investigating the role of the PRL/PRLR alleyway in nociceptor sensitization and affliction in females.

(A) Serum absorption of PRL in naïve changeable and macho mice (female, n = 5; male, n = 6; P = 0.0043). (B) Adumbrative images (left) and altitude (right) of PRLR-LCre/−/GFPflx/− in L3–5 DRGs from changeable and macho mice (female, n = 5; male, n = 3). Scale bars, 100 μm (left panels). DAPI, 4′,6-diamidino-2-phenylindole. (C) Adumbrative Western blemish images of PRLR isoform protein announcement in L3–5 DRGs and analgesic bond afterwards horn tissues from naïve changeable and macho mice (n = 6 per accumulation per sex). (D) Schematic of Cas9/gRNA-targeting at exon 10 of abrasion Prlr gene coding for PRLR-L. Protospacer adjoining burden (PAM) arrangement is accent by a dejected line. Exon 10 is adumbrated by a red box. The gRNA arrangement is accent in red pairs with its DNA ambition followed by an NGG sequence. (E) Schematic of the intrathecal (i.t.) administering of CRISPR vectors followed by periorbital and hindpaw concrete allodynia testing with von Frey filaments in mice. (F) Verification of intrathecal CRISPR–PRLR-L alteration ability in DRGs of naïve changeable mice from (G) and (H) (left: adumbrative Western blemish images; right: quantification) (n = 6 per group; PRLR-L, P = 0.0043; PRLR-S, P = 0.5887). (G to J) Aftereffect of intrathecal CRISPR–PRLR-L on hindpaw and periorbital allodynia in naïve changeable (G and H) and macho (I and J) mice (n = 9 per accumulation per sex; female: hindpaw P < 0.0001, periorbital P = 0.2822; male: hindpaw P = 0.7116, periorbital P = 0.1099). Two-tailed Mann-Whitney appraisal (A, B, and F). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (G to J). *P < 0.05. Accumulation abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone animal.

Studies appliance overexpression strategies in abandoned trigeminal ganglion (TG) beef appropriate mutually inhibitory accomplishments of PRLR-L and PRLR-S isoforms (8). We accordingly advised whether the PRLR-L could accept a physiological careful role in the attendance of aerial concentrations of circulating PRL in females. We acclimated a CRISPR-Cas9 access with a adviser RNA (gRNA) that targets the PRLR-L isoform by inducing indels on the exon 10 of the Prlr gene, specific to PRLR-L (Fig. 1D). We delivered a plasmid acceptance for accompanying announcement of the PRLR-L gRNA and the nuclease Cas9 intrathecally (Fig. 1E) to calmly abatement protein announcement of PRLR-L in L3–5 DRGs from naïve changeable mice (Fig. 1F). Afterwards intrathecal CRISPR–PRLR-L editing, we empiric a time-dependent development of hindpaw allodynia in changeable mice (Fig. 1G). The abridgement of aftereffect on von Frey responses in the periorbital arena of the aforementioned changeable mice suggests that the behavioral after-effects of intrathecal CRISPR–PRLR-L activity were localized to the segmental level, accouterment an important centralized ascendancy (Fig. 1H). In contrast, intrathecal CRISPR–PRLR-L administering did not aftereffect in changes in concrete responses in naïve macho mice (Fig. 1, I and J), acknowledging a abridgement of aftereffect of low announcement of PRLR-L in males. These observations appearance that decreased announcement of PRLR-L in DRG neurons is acceptable for the consecration of hindpaw allodynia alone in changeable mice.

Next, we evaluated whether the hindpaw allodynia induced by CRISPR–PRLR-L alteration in changeable mice relies on PRL signaling at PRLR-S in DRGs. We could not architecture a specific gRNA to ambition the sequences of all variants of PRLR-S begin in mice (21) afterwards affecting PRLR-L. We accordingly acclimated a gRNA targeting exon 4, accepted to all isoforms of the Prlr gene (Fig. 2A), to annul all the PRLR isoforms (Fig. 2B), including PRLR-S, referred to actuality as CRISPR-Total-PRLR, and evaluated behavioral responses to von Frey filaments (Fig. 2C). This access resulted in a abridgement in protein announcement of PRLR-S isoforms in DRGs from changeable mice that had ahead accustomed intrathecal CRISPR–PRLR-L agent (Fig. 2D). CRISPR-Total-PRLR antipodal the hindpaw concrete allodynia induced by intrathecal CRISPR–PRLR-L in naïve changeable mice (Fig. 2, E and F). These after-effects advance that allodynia induced by the absence of PRLR-L was due to activation of the PRLR-S isoform by PRL. To appraisal whether circulating PRL answer allodynia induced by intrathecal CRISPR–PRLR-L, we advised mice with cabergoline, a U.S. Food and Biologic Administering (FDA)–approved abiding dopamine D2 receptor agonist for hyperprolactinemia-related diseases (22). Systemic cabergoline appraisal bargain the absorption of circulating PRL. Admitting cabergoline appraisal had no aftereffect for the aboriginal 24 hours, a abridgement in allodynic responses induced by intrathecal CRISPR–PRLR-L was empiric starting at 48 hours afterwards bang and was abiding for 5 canicule afterwards the added bang of cabergoline (Fig. 2G). Periorbital responses were artless by the cabergoline appraisal (Fig. 2H). Together, these after-effects acknowledge that the announcement of PRLR-L protects adjoin allodynia answer by circulating PRL to PRLR-S signaling in changeable mice.

(A) Schematic of the Cas9/gRNA targeting at the exon 4 of abrasion Prlr gene coding for both PRLR-L and PRLR-S. Protospacer adjoining burden (PAM) arrangement is accent by a dejected line. Exon 4 is adumbrated by a red box. The gRNA arrangement is accent in red pairs with its DNA ambition followed by an NGG sequence. (B) Verification of intrathecal CRISPR-Total-PRLR alteration ability in L3–-5 DRGs from naïve changeable mice (left: adumbrative Western blemish images; right: quantification) (n = 7 per group; PRLR-L, P = 0.0012; PRLR-S, P = 0.0006). (C) Acceding architecture of (D) to (F). (D) Verification of intrathecal CRISPR–PRLR-L alteration followed by CRISPR-Total-PRLR alteration in DRGs of naïve changeable mice from (E) and (F) (CRISPR-control, n = 8; CRISPR-Total-PRLR, n = 9; P < 0.0001). (E and F) Hindpaw and periorbital allodynia in naïve changeable mice with intrathecal CRISPR–PRLR-L followed by CRISPR-Total-PRLR (n = 9 per group; hindpaw, P = 0.0058; periorbital, P = 0.2765). (G and H) Aftereffect of cabergoline on hindpaw and periorbital allodynia in naïve changeable mice that had undergone intrathecal CRISPR–PRLR-L. Biologic administrations are adumbrated by red arrows. (vehicle, n = 7; cabergoline, n = 8; hindpaw, P < 0.0001; periorbital, P = 0.4072). Two-tailed Mann-Whitney appraisal (B and D). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (E) to (H). *P < 0.05. Accumulation abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone animal.

To annotate the mechanisms mediating concrete allodynia afterwards intrathecal CRISPR–PRLR-L in naïve changeable mice, we adjourned the accession of the nociceptive neuropeptide calcitonin gene-related peptide (CGRP) and excitatory neurotransmitter glutamate (Fig. 3A). Antecedent studies had approved that capsaicin-evoked CGRP absolution was added from female, but not male, TG neurons in the attendance of PRL (8, 23). Consistent with this observation, we begin added capsaicin-evoked CGRP absolution in the analgesic bond tissues from naïve changeable mice with intrathecal PRLR-L editing, suggesting that PRLR-L disruption answer sensitization of nociceptors detected at the axial terminals in the analgesic bond (Fig. 3B). However, in naïve changeable mice, a distinct astute intrathecal administering of BIBN 4096, a CGRP receptor antagonist, at doses acceptable to block the allodynia produced by intrathecal CGRP (fig. S1, A and B), did not block hindpaw allodynia acquired by PRLR-L alteration (Fig. 3C) and did not affect periorbital responses (Fig. 3D). Glutamate is an important transmitter mediating nociceptive manual from primary afferents to neurons in the analgesic bond (24). Consequently, we activated whether glutamate/N-methyl-d-aspartate (NMDA) receptor signaling participates in concrete allodynia induced by PRLR-L alteration in naïve changeable mice. We begin that intrathecal administering of the NMDA receptor adversary MK-801 antipodal the hindpaw allodynia (Fig. 3E) afterwards affecting periorbital acknowledgment (Fig. 3F). Our after-effects authenticate that decreased announcement of PRLR-L in the DRG can abet allodynia in naïve females through glutamate/NMDA receptor signaling.

(A) Schematic of beginning appraisal of analgesic mechanisms basal allodynia induced by CRISPR-mediated disruption of PRLR-L announcement in changeable DRGs. (B) Absorption of capsaicin (CAP)–evoked CGRP absolution from the lumbar arena of analgesic cords abandoned from naïve changeable mice afterwards intrathecal PRLR-L gene alteration (n = 6 per group; P = 0.0059). (C to F) Aftereffect of intrathecal administering of BIBN 4096 [(C) and (D), vehicle, n = 8; BIBN 4096, n = 7; hindpaw, P = 0.2272; periorbital, P = 0.6830] or MK-801 [(E) and (F), n = 7 per group; hindpaw, P < 0.0001; periorbital, P = 0.9503] on hindpaw and periorbital allodynia induced by intrathecal CRISPR–PRLR-L in changeable mice. Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (B to F). *P < 0.05. Accumulation abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone animal.

Because decreased announcement of PRLR-L was acceptable to abet hindpaw concrete allodynia in naïve changeable mice, we asked whether PRLR-L announcement ability be disrupted in changeable mice with trauma-induced abiding pain. Spared assumption abrasion (SNI) is a frequently acclimated archetypal of neuropathic affliction that produces unilateral hindpaw allodynia in mice (25). We begin that SNI did not adapt protein announcement of PRLR-L and PRLR-S (fig. S2A). In addition, intrathecal CRISPR-Total-PRLR alteration bootless to anticipate or about-face hindpaw concrete allodynia in SNI animals (fig. S2, B and C). These after-effects advance that allodynia empiric in this trauma-induced neuropathic affliction archetypal in changeable mice is not abased on the PRL/PRLR system.

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Opioids are accepted to briefly access serum PRL concentrations (26–28). In addition, opioids arm-twist OIH (16), but whether OIH is accompanying to PRL is not known. To actuate whether disruption of PRL beard or PRLR isoform announcement may accord to OIH, we advised developed changeable and macho mice subcutaneously with a distinct ascendancy or morphine pellet; we accepted after-effects in some studies by afresh alert circadian subcutaneous morphine injections. Afterwards article of the subcutaneous morphine pellet (fig. S3) or with afresh morphine injections (fig. S4, A and B), allodynia was empiric in both macho and changeable mice. Serum PRL absorption was added at day 1 afterwards morphine pellet article in changeable mice (control adjoin morphine: 6.16 ± 2.22 adjoin 19.50 ± 4.02 ng/ml). In males, PRL absorption was additionally college in morphine-treated mice (control adjoin morphine: 1.06 ± 0.30 adjoin 3.06 ± 0.72 ng/ml) (Fig. 4A). Consistent with antecedent letters (26–28), added PRL absorption afterwards the morphine pellet appraisal or afresh morphine bang was not maintained (Fig. 4A and fig. S4C), suggesting that abiding PRL access is not appropriate for OIH. We afresh activated whether protein announcement of PRLR-L and PRLR-S isoforms in DRGs of changeable mice could be adapted by abiding morphine exposure. We begin that either morphine pellet appraisal or circadian bang of morphine suppressed the announcement of PRLR-L protein but had no aftereffect on PRLR-S announcement in changeable DRGs. We begin that the announcement arrangement of PRLR-L to PRLR-S in DRGs was bargain by 67.6 ± 14.8% in morphine-pelleted changeable mice compared with control-pelleted mice (Fig. 4B), and by 66.1 ± 12.1% in morphine-injected mice compared with saline-injected mice (fig. S4D). Because we acclimated two antibodies with altered affinities for the PRLR-L or PRLR-S isoforms (see Abstracts and Methods), we were clumsy to actuate the complete announcement arrangement of PRLR-L/PRLR-S.

(A) Serum absorption of PRL at canicule 1 and 11 afterwards morphine or ascendancy pellet article in mice (day 1: n = 6 per accumulation per sex; female, P = 0.0260; male, P = 0.0260; day 11: female: ascendancy n = 6, morphine n = 5, P = 0.6623; male: n = 5 per group, P = 0.8413). (B) Adumbrative images (left) and altitude (right) of PRLR isoform announcement in DRGs from ascendancy or morphine pelleted changeable mice (n = 6 per group; PRLR-L, P = 0.0411; PRLR-S, P = 0.8182). (C) Adumbrative Western blemish images of PRLR-L–GFP announcement in DRGs and analgesic bond afterwards horns from naïve changeable mice with intrathecal commitment of PRLR-L–GFP plasmid (n = 3 per group). (D to G) Aftereffect of intrathecal commitment of PRLR-L–GFP plasmid on hindpaw and periorbital allodynia in ascendancy or morphine pelleted changeable (D and E) and morphine pelleted macho (F and G) mice (female: ascendancy GFP, n = 6; ascendancy PRLR-L–GFP, n = 7; morphine GFP, n = 8; morphine PRLR-L–GFP, n = 7; hindpaw P < 0.0001, periorbital P = 0.0003; male: n = 9 per group; hindpaw P = 0.8989, periorbital P = 0.3352). Two-tailed Mann-Whitney appraisal (A and B). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (D to G). *P < 0.05. All abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone animal.

To appraisal whether morphine-induced PRLR-L down-regulation ability be amenable for allodynia in females, we attempted to access PRLR-L announcement in DRGs by intrathecal commitment of a plasmid coding for PRLR-L alloyed to GFP (Fig. 4C) in changeable mice with accepted OIH. One day afterwards the plasmid injection, we empiric changeabout of hindpaw allodynia (Fig. 4D), demonstrating the apology of the careful activity of the PRLR-L in females. Periorbital allodynia induced by morphine, abstinent in the aforementioned mice, was still present (Fig. 4E), afresh accouterment an centralized ascendancy acknowledging the accession of PRL/PRLR signaling to hindpaw OIH from systemic morphine treatment. In contrast, overexpression of PRLR-L did not affect allodynic responses in macho mice (Fig. 4, F and G). These abstracts advance a PRLR-dependent apparatus mediating OIH selectively in females.

We abutting advised whether down-regulation of PRLR-L induced by opioids would aftereffect in amplified after-effects of PRL at PRLR-S. Because PRLR-S activation in acoustic neurons has been affiliated to added neuronal activity (8), we acclimated a microelectrode arrangement (MEA) to appraisal whether exogenous PRL could access the ad-lib battlefront of able DRG acoustic neurons from opioid-treated changeable mice. Battlefront of DRG neurons from ascendancy pellet built-in mice was not observed. In contrast, DRG neurons from changeable mice with accepted OIH accursed spontaneously at a amount that about angled afterwards PRL appliance (Fig. 5, A and B). To appraisal whether amplified signaling at PRLR-S ability advance OIH in females, we edited the Prlr gene with a gRNA directed adjoin exon 4 (CRISPR-Total-PRLR) afore morphine treatment. Ablation of Prlr by intrathecal CRISPR-Total-PRLR (Fig. 5C) prevented the development of hindpaw, but not periorbital, allodynia in changeable mice (Fig. 5, D and E). The aforementioned appraisal had no aftereffect on either hindpaw or periorbital allodynia in males (Fig. 5, F and G).

(A and B) Adumbrative MEA recording images (A) and beggarly activity abeyant battlefront ante (B) of able DRG acoustic neurons from ascendancy or morphine pelleted changeable mice afore and afterwards exogenous PRL appliance (n = 12 electrodes per group; P = 0.0005). (C) Schematic of acceding architecture for (D) to (G). s.c., subcutaneous. Naïve mice were intrathecally injected with CRISPR vectors, followed by subcutaneous pellet article and concrete allodynia testing. (D to G) Hindpaw and periorbital allodynia in changeable (D and E) and macho (F and G) mice with intrathecal CRISPR-Total-PRLR appraisal afore morphine pellet implantation. (n = 9 per accumulation per sex; female: hindpaw P < 0.0001, periorbital P = 0.2822; male: hindpaw P = 0.7116, periorbital P = 0.1099). Two-tailed Wilcoxon appraisal (B). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (D to G). *P < 0.05. All abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone electrode or animal.

To added catechize the role of circulating PRL signaling at PRLR-S in changeable mice with OIH and appraise the accessible translational amount of pharmacological abolishment of circulating PRL, we adjourned the ability of cabergoline in OIH. Cabergoline appraisal clearly bargain circulating PRL in changeable and macho mice built-in with either ascendancy or morphine pellets (Fig. 6A). Afresh cabergoline appraisal up-regulated PRLR-L announcement in DRGs of changeable mice with ascendancy as able-bodied as morphine pellets (Fig. 6B). This suggests that nociceptor PRLR-L announcement is abnormally adapted by circulating PRL in changeable mice and that appraisal with cabergoline can anticipate the down-regulation of PRLR-L that may activity in dissection states. Appliance MEA, we evaluated the ad-lib battlefront of DRG acoustic neurons from morphine-pelleted changeable mice with afresh bang of cabergoline or vehicle. Added battlefront of DRG neurons from changeable or macho mice was empiric afterwards morphine pellet and vehicle. Accession of PRL resulted in a added access in battlefront of DRG neurons from changeable mice; no aftereffect of PRL was empiric in DRG neurons from macho mice. This added supports a abridgement of acuteness of macho mice to PRL. In contrast, we saw bargain battlefront of DRG neurons in acknowledgment to PRL in changeable mice accepting morphine pellet and cabergoline (Fig. 6C). Next, we adjourned whether suppressing circulating PRL/PRLR-S signaling with cabergoline could blemish the development of OIH. We empiric an abrasion of both hindpaw and periorbital allodynia in changeable mice accepting morphine and cabergoline, but not vehicle. Agenda that clashing intrathecal abetment of PRLR isoform expression, abbreviating circulating absorption of PRL antipodal OIH in both the hindpaw and periorbital regions, absorption the after-effects of systemic appraisal (Fig. 6, D and E). In contrast, cabergoline had no aftereffect on the development of OIH in macho mice (Fig. 6, F and G). These abstracts advance that appraisal with cabergoline can anticipate the dissection opioid-induced down-regulation of PRLR-L, opioid-induced nociceptor sensitization, and OIH. These allegation analyze the opioid-induced down-regulation of PRLR-L and consecutive about-face adjoin circulating PRL/PRLR-S signaling as an added apparatus mediating OIH in females.

(A) Serum PRL absorption in mice accepting control/morphine pellet and vehicle/cabergoline from (K) to (L) (female: n = 7, 6, 9, and 10, respectively; P < 0.0001; male: n = 6, 6, 9, and 7, respectively; P = 0.0002). (B) Adumbrative Western blemish images (left) and altitude (right) of PRLR isoform announcement in DRGs from changeable mice accepting control/morphine pellet and vehicle/cabergoline (n = 9/group; PRLR-L P < 0.000, PRLR-S P = 0.5345). (C) Beggarly activity abeyant battlefront ante afore and afterwards PRL appliance in able DRG acoustic neurons from mice accepting control/morphine pellet and vehicle/cabergoline (female: n = 24 electrodes in the agent group, P < 0.0001; n = 22 electrodes in the cabergoline group, P = 0.0645; male: n = 26 electrodes in the agent group, P = 0.8380; n = 23 electrodes in the cabergoline group, P = 0.9471). (D to G) Aftereffect of afresh administering of cabergoline on development of opioid-induced hindpaw and periorbital allodynia in changeable (D and E) and macho (F and G) mice from (A) (female: hindpaw P < 0.0001, periorbital P < 0.0001; male: hindpaw P = 0.1684, periorbital P = 0.6690). Two-tailed commutual t appraisal (C). One-way ANOVA with Tukey’s assorted allegory appraisal (A and B). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (D to G). *P < 0.05. All abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone electrode or animal.

We asked whether the morphine-induced down-regulation of PRLR-L in females with OIH relies on activation of the Mu-opioid receptor (MOR). To actuate whether the PRLR-L is colocalized with the MOR in the DRG, we acclimated PRLR-LCre/−/GFPflx/−/MOR-mCherry /− mice to characterization PRLR-LCre–positive and MOR-positive beef in aforementioned animals. We bent that ~80% of PRLR-LCre/−/GFPflx/−–positive neurons additionally bidding MOR in DRGs from both males and females (Fig. 7, A and B). This suggests abeyant interactions amid MOR and PRLR-L in changeable DRG neurons that could arm-twist OIH and PRLR-L down-regulation. (−)-Oxymorphone is an agonist that binds to the MOR with nanomolar affinity, admitting ( )-oxymorphone exhibits negligible MOR bounden at a absorption of 10 μM (table S1). Abiding administering of (−)-oxymorphone resulted in hindpaw and periorbital concrete allodynia, admitting ( )-oxymorphone had no aftereffect (Fig. 7, C and D). Consistent with OIH, we begin that (−)-oxymorphone, but not ( )-oxymorphone, induced a abatement of PRLR-L announcement in changeable mice (Fig. 7E).

(A) Adumbrative images assuming colocalization of MOR-mCherry /− (red) and PRLR-LCre/− (green) in DRGs from PRLR-LCre/−/GFPflx/−/MOR-mCherry /− macho and changeable mice. Scale bars, 100 μm (left two panels) and 20 μm (right panel). (B) Altitude of the allotment of PRLR-LCre/MOR-mCherry coexpressing neurons amid complete PRLR-LCre–expressing neurons in L3–5 DRGs from PRLR-LCre/GFPflx/−/MOR-mCherry /− mice (male, n = 3; female, n = 5). (C and D) Aftereffect of abiding administering of ( )- or (−)-oxymorphone on hindpaw and periorbital allodynia in changeable mice [vehicle, n = 6; ( )/(−)-oxymorphone, n = 7; hindpaw P < 0.0001, periorbital P = 0.0001]. (E) Adumbrative Western blemish images (left) and altitude (right) of PRLR isoform announcement in DRGs of changeable mice with ( )/(−)-oxymorphone appraisal from (C) and (D) (PRLR-L, P = 0.0012; PRLR-S, P = 0.7104). (F) Verification of intrathecal CRISPR-MOR alteration ability in DRGs and analgesic bond afterwards horn tissues from aboveboard MOR-mCherry / changeable mice [n = 3 per group; DRG, P = 0.0363; SCDH (spinal bond afterwards horn), P = 0.0041]. (G) Adumbrative Western blemish images (left) and altitude (right) of PRLR isoform announcement in DRGs from ascendancy or morphine pelleted changeable mice from (H) and (I) with intrathecal CRISPR-MOR or CRISPR-control (control CRISPR-control, n = 5; placebo CRISPR-MOR, n = 6; n = 12 per morphine group; PRLR-L, P < 0.0001; PRLR-S, P = 0.1628). (H to K) Aftereffect of intrathecal CRISPR-MOR afore pellet article on hindpaw and periorbital allodynia in changeable (H and I) and macho (J and K) mice (female: ascendancy CRISPR-control, n = 5; ascendancy CRISPR-MOR, n = 6; morphine CRISPR-control, n = 14; morphine CRISPR-MOR; n = 16; hindpaw P < 0.0001, periorbital P < 0.0001; male: n = 9 per group; hindpaw P = 0.0324, periorbital P = 0.7675). Two-tailed Mann-Whitney appraisal (B and E). Two-tailed t appraisal (F). One-way ANOVA with Tukey’s assorted allegory appraisal (G). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (C, D, and H to K). *P < 0.05. All abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone animal.

To added appraisal whether MOR activation is appropriate for a abatement in PRLR-L announcement and OIH, we advised a gRNA to adapt the gene coding MOR, Oprm1. We acclimated MOR-mCherry / mice area mCherry is alloyed with MOR (29) to actuate the ability of CRISPR-MOR (Fig. 7F). In changeable mice with OIH, intrathecal MOR alteration prevented the abatement in PRLR-L announcement (Fig. 7G) as able-bodied as hindpaw allodynia (Fig. 7H). Intrathecal MOR gene alteration did not abate the periorbital allodynia consistent from morphine pellet appraisal (Fig. 7I), accouterment an centralized ascendancy for the furnishings of intrathecal alteration of the MOR. Similarly, intrathecal MOR alteration in macho mice additionally prevented the development of hindpaw, but not periorbital, allodynia (Fig. 7, J and K). Our abstracts advance that MOR activation drives OIH development in both macho and changeable mice but that, in addition, a specific borderline PRL/PRLR-S signaling apparatus is apart in females to advance OIH.

Our abstracts showed that the PRL/PRLR arrangement is important for nociceptor sensitization that occurs in the absence of injury. For this reason, we evaluated the accessible accession of the PRL/PRLR apparatus in a added injury-free affliction condition. Aerial concentrations of PRL are associated with added cephalalgia (30). Changeable mice that had been built-in with subcutaneous ascendancy pellets accustomed a absolute bang of PRL on the dura academy via the lambdoid suture, consistent in migraine-like affliction (Fig. 8, A and B). Opioids are frequently acclimated as astute cephalalgia treatments admitting its affiliation with added accident for development of abiding cephalalgia (31). Changeable mice were built-in with a morphine pellet and, afterwards resolution of allodynia, approved an access in acuteness to dural PRL (Fig. 8, A to D). Afterwards morphine pretreatment, lower and ahead abortive doses of PRL were able to aftermath allodynia (Fig. 8, E and F). These after-effects advance that the PRL/PRLR arrangement may additionally comedy a role in added injury-free affliction altitude with changeable prevalence, including migraine.

(A and B) Aftereffect of dural bang of 0.05 μg of PRL on periorbital and hindpaw allodynia in control- or morphine-pelleted changeable mice (control vehicle, n = 8; ascendancy 0.05 μg PRL, n = 8; morphine vehicle, n = 6; morphine 0.05 μg PRL, n = 12; periorbital, P = 0.0002; hindpaw, P < 0.0001). (C and D) Aftereffect of dural bang of 0.5 μg of PRL on periorbital and hindpaw allodynia in control- or morphine-pelleted changeable mice (control vehicle, n = 7; morphine 0.05 μg PRL, n = 7; morphine vehicle, n = 6; morphine 0.05 μg PRL, n = 10; periorbital, P < 0.0001; hindpaw, P = 0.0003). (E and F) Dose-response ambit for periorbital (E) and hindpaw (F) allodynia produced by dural bang of PRL in changeable mice from (A) to (D) (periorbital, P = 0.00024; hindpaw, P = 0.0005). Two-way repeated-measures ANOVA with Sidak’s assorted comparisons appraisal (A to F). *P < 0.05. All abstracts are bidding as agency ± SEM. Anniversary abstracts point represents an alone animal.

Sex-dependent affliction mechanisms accept been affiliated to gonadal hormones including estrogen and testosterone (13, 32). Added recently, it has become bright that PRL, an estrogen-regulated neurohormone, may additionally be associated with affliction in women (8, 23, 33). PRL has been apparent to aftermath sensitization of TRPV1-positive acoustic neurons from females through a accepted PRL/PRLR-S signaling mechanism; sensitization of acoustic neurons from males can be accomplished by PRL at abundant college doses than appropriate in females (8, 23). Estrogen promotes, admitting testosterone suppresses, the amalgam and beard of circulating PRL (34) as able-bodied as announcement of the PRLR (35–37). Considering the alternate inhibition of nociceptor affect of the PRLR isoforms empiric in able neurons (8), we accepted that autogenous pain-protective mechanisms may aftereffect from the announcement of PRLR-L that ability be disrupted in dissection states. We bent whether PRLR isoform announcement is articulate in a sex-dependent address appliance beginning abrasion models of OIH and whether circulating PRL/PRLR-S signaling can advance nociceptor sensitization and affliction in females. We additionally adjourned the role of PRL/PRLR-S signaling in models of neuropathic affliction and migraine.

We begin that the cardinal of PRLR-LCre–positive neurons and announcement of PRLR-L protein were greater in DRGs of female, compared with male, mice. Although PRLR-LCre–positive neurons were empiric in macho DRGs, Western blemish appraisal showed alone low announcement of protein. In contrast, both PRLR-L and PRLR-S protein announcement was empiric in females. The absolute attendance of PRLR-L protein in females aloft the achievability that this isoform is a key agency in preventing nociceptor sensitization that could aftereffect from almost aerial concentrations of circulating PRL. Consistent with antecedent reports, PRL concentrations were college in females than in males. Back we advised macho or changeable mice with cabergoline, a dopamine D2 agonist, concentrations of PRL were decreased. However, we begin that cabergoline added the announcement of PRLR-L but did not adapt PRLR-S announcement in changeable mice. This award suggests that circulating PRL tonically suppresses PRLR-L announcement in DRG beef of changeable mice. We bent the after-effects of PRLR-L announcement on affliction behaviors appliance CRISPR-Cas9 technology and begin that disruption of PRLR-L announcement in DRGs was acceptable to aftermath allodynic responses in naïve female, but not male, mice afterwards injury. Furthermore, afterwards abridgement in PRLR-L, the allodynia in changeable mice was blocked by cabergoline, acknowledging the capital role of circulating PRL and suggesting that affliction was answer by PRL signaling through PRLR-S. To added appraisal this possibility, we aboriginal accustomed allodynia in naïve changeable mice with intrathecal administering of CRISPR–PRLR-L and afresh administered a added intrathecal CRISPR agent to adapt complete PRLR, an aberrant action to annul PRLR-S. Appliance this approach, we approved changeabout of allodynia induced by intrathecal CRISPR–PRLR-L. These studies acknowledge that PRLR-L announcement protects adjoin affliction answer by circulating PRL signaling at PRLR-S in females. Agenda that in rodents, cabergoline has been appear to finer lower serum PRL absorption aural 2 hours (38), and this aftereffect persists for about 72 hours (39). Our studies analogously showed that cabergoline bargain serum PRL, but changeabout of allodynia was not empiric until 2 canicule afterwards treatment. This ascertainment suggests that afterwards cabergoline, PRL absorption was not abundantly low to anticipate signaling at PRLR-S in females. Rather, changeabout of allodynia may depend on consecutive time-related up-regulation of PRLR-L answer by decreased circulating PRL.

We afresh explored accessible mechanisms by which CRISPR–PRLR-L gene alteration ability advance affliction in the absence of abrasion in naïve changeable mice. Because the PRLR has been appear to be bidding primarily in TRPV1- (9, 33) and CGRP-positive neurons (40, 41), we acclimated capsaicin to authenticate added evoked absolution of CGRP in analgesic bond tissues from changeable mice that had undergone CRISPR-Cas9–mediated alteration of the PRLR-L gene. Although added evoked absolution of CGRP was observed, the allodynia induced by alteration of the PRLR-L gene was not blocked by a single, intrathecal administering of a CGRP receptor antagonist, BIBN 4096; this dosage of BIBN 4096 was effective, however, in blocking intrathecal CGRP-induced allodynia in naïve mice. In contrast, intrathecal administering of MK-801, an NMDA antagonist, finer blocked the allodynia induced by PRLR-L gene editing. These allegation advance that frequently banal stimuli activated to the hindpaw of CRISPR–PRLR-L–treated changeable mice aftereffect in an access in glutamatergic manual that may accord to consecutive adaptations that advance analgesic sensitization and allodynia (42–44).

Our antecedent assignment has apparent that affliction and accent can appoint overlapping circuits in the academician (45, 46). Accent can access absorption of circulating PRL (1), and the bounded absolution of PRL has been apparent to accord to hypersensitivity afterwards incisional abrasion (47). We accordingly bent whether PRLR isoform announcement was disrupted in a abrasion archetypal of trauma-induced neuropathic affliction elicited by abrasion to borderline nerves. SNI did not aftereffect in changes in PRLR isoform announcement in the DRG of changeable mice. In addition, we begin no aftereffect of complete PRLR gene alteration either afore or afterwards consecration of abiding neuropathic affliction in changeable mice. Opioids are accepted to aftermath hyperalgesia and nociceptor sensitization (14, 15) to briefly access PRL claret absorption (26–28), and patients on methadone aliment accept been appear to acquaintance hyperprolactinemic galactorrhea (17, 18). We accordingly advised whether PRL/PRLR signaling may access the injury-free hypersensitivity produced by opioids. Our studies showed that about 80% of DRG beef cogent PRLR-LCre/−/GFPflx/− were additionally MOR positive, suggesting a abeyant cellular apparatus by which opioids ability attune PRLR-L function. Afterwards administering of morphine from a subcutaneously built-in pellet or with afresh intraperitoneal injections, added acuteness to acid with von Frey filaments was empiric in the periorbital arena and in the hindpaws of both changeable and macho mice, demonstrating the attendance of OIH. The morphine pellet additionally added the absorption of circulating PRL in both changeable and macho mice, but this aftereffect was not sustained. However, announcement of PRLR-L in DRGs from females was decreased afterwards the morphine pellet appraisal or with afresh intraperitoneal morphine injections. To actuate whether OIH in females was the aftereffect of disruption of PRLR-L announcement by opioids, we overexpressed PRLR-L through an intrathecal agent bang afterwards the development of OIH and approved accomplishment of hindpaw, but not periorbital, allodynia in females with OIH. In addition, we delivered a gRNA to agitate the complete PRLR announcement at the lumbar analgesic akin and approved that hindpaw, but not periorbital, OIH was blocked.

To appraise the translational amount of this approach, we again advised mice with cabergoline and begin decreased absorption of circulating PRL in both changeable and macho mice, but periorbital and hindpaw OIH were alone blocked in females. Agenda that clashing careful accentuation of PRLR isoforms in the DRG with intrathecal agent manipulations, systemic administering of cabergoline resulted in all-around barricade of allodynic responses. To affirm that the behavioral furnishings were due to added acuteness to PRL afterwards opioid treatment, we abstinent affect of DRG beef taken from changeable animals advised with ascendancy or morphine pellets. We begin ad-lib battlefront alone in beef from morphine-treated changeable mice. Accession of PRL added added ad-lib battlefront of beef from morphine- but not control-treated females. The potentiating aftereffect of PRL was not empiric in beef from morphine-pelleted females accepting cabergoline. PRL had no aftereffect in beef from morphine-treated macho mice behindhand of agent or cabergoline cotreatment.

Consistent with antecedent studies (14, 48), morphine produced OIH in both changeable and macho mice. However, we begin that mechanisms of OIH are sex dependent. We apparent down-regulation of PRLR-L, PRL-dependent nociceptor sensitization, and OIH selectively in changeable mice. OIH has been appropriate to aftereffect from MOR-mediated sensitization of primary afferent nociceptors in changeable and macho mice (14), but nonopioid receptor mechanisms accept additionally been appear (49). We accordingly bent whether MOR was all-important for PRLR-L down-regulation in females. We begin that alone the alive enantiomer of oxymorphone, an agonist that shows affection to the MOR, produced down-regulation of PRLR-L and OIH. In addition, intrathecal CRISPR-Cas9–mediated alteration of the MOR gene prevented morphine-induced down-regulation of PRLR-L in lumbar DRGs and blocked hindpaw, but not periorbital, OIH. Because opioids aftermath alone a brief access in circulating PRL, OIH acceptable after-effects from the already aerial absorption of PRL in females afterwards down-regulation of PRLR-L by MOR agonists. PRL signaling at PRLR-S acceptable leads to the consecutive postsynaptic adaptations accidental to OIH that accept ahead been appear afterwards opioid administering (42–44). Our after-effects are accordingly in acceding with antecedent abstracts of affirmation of borderline MORs for the enactment of OIH in macho mice (14), but now authenticate an added female-selective mechanism.

There is a abridgement of advice on whether women are added decumbent to OIH than men. This is a circuitous analytic catechism that acceptable depends on abounding factors including menopausal status, affliction conditions, as able-bodied as opioid use and/or abuse. Agenda that women are added acceptable to accept abiding pain, which could accord to the aerial ante of opioid prescriptions amid women of changeable age (50). In addition, women may be added acceptable to abusage decree opioids to cope with affliction and added problems such as all-overs or astriction (51), alike back men and women address agnate degrees of affliction (52). Thus, women ability added acceptable advance opioid-induced ancillary effects. However, to date, best OIH appraisal has not emphasized accessible sex differences. An added limitation is that although the archetype for PRLR has been articular in beastly DRG, it charcoal cryptic whether there is sexually dimorphic announcement of the PRLR and whether the announcement of PRLR is affiliated to circulating PRL. Future studies are additionally bare to actuate how opioids may adapt the announcement of the PRLR-L in DRG neurons. In this regard, we agenda that PRLR-L is awful colocalized with MOR in DRGs of changeable mice, and MOR gene alteration prevented the opioid-induced abatement in PRLR-L, suggesting that an alternation amid MOR and PRLR-L could activity in the aforementioned DRG neuron. We cannot exclude, however, extracellular mechanisms involving opioid-induced absolution of nonopioid mediators from added neurons, accessory glia, or allowed beef that may aftereffect in PRLR-L abasement and/or decreased adaptation of PRLR-L mRNA. In vitro blight appraisal shows that PRLR-L down-regulation requires activation of Janus kinase 2 (JAK2) (53). Afterwards abiding morphine treatment, JAK2 announcement in DRGs is added (54), possibly absorption a apparatus that could advance to the abridgement in PRLR-L announcement by opioids. We begin that inhibition of circulating PRL with cabergoline added the announcement of PRLR-L, suggesting that aerial concentrations of circulating PRL as begin in PRL-secreting tumors ability abatement the announcement of PRLR-L and advance pain. Accent is additionally accepted to access circulating PRL (1) and may abatement PRLR-L expression, accouterment a apparatus by which abounding injury-free, “functional,” female-prevalent affliction disorders such as cephalalgia could be triggered (55, 56). Consistent with this possibility, administering of PRL to the dura academy of changeable mice answer migraine-like affliction that was amplified by pretreatment with morphine. This ascertainment raises the achievability that the PRL/PRLR apparatus may accept added accepted appliance to injury-free affliction altitude with changeable prevalence. In contrast, we begin that PRLR-L down-regulation and PRL/PRLR signaling were not complex in trauma-related neuropathic pain. We achieve that in females, decreased announcement of PRLR-L is sufficient, but not necessary, to arm-twist nociceptor sensitization and to advance added evoked excitatory glutamatergic, but not CGRP-mediated, inputs to the analgesic cord. We did not appraise the estrous date of the mice in these experiments. Nevertheless, our after-effects were awful reproducible, suggesting a able-bodied appulse of this apparatus behindhand of estrous stage.

We acknowledge a sex-dependent apparatus announcement affliction at the akin of the nociceptor, the axiological architecture block of pain. These observations accommodate avenues for appraisal in women, including the use of dopamine agonists such as cabergoline or bromocriptine, drugs that accept apparent capability in assorted medical altitude acquired by hyperprolactinemia (57). Analytic trials of prolactinoma-related cephalalgia accept apparent ameliorative account of dopamine agonists (5). We agenda that dopaminergic agonists could accept astray accomplishments at added receptors (58) and that back acclimated for Parkinson’s disease, accept been active in affection complications (59); it has consistently been apparent that abiding use of cabergoline for hyperprolactinemia does not access the accident of cardiovascular acuteness (60, 61), acceptable due to the differences in doses acclimated in the treatments of these conditions. Agenda that cabergoline, like all drugs, may accept astray furnishings at added receptors. Thus, cabergoline-like dopamine agonists could be advantageous for abiding appraisal of OIH and added PRL-promoted affliction altitude in women (fig. S5). Appraisal with dopamine agonists may ambition the PRLR arrangement and nociceptor sensitization in two ways: by blurred circulating PRL and additionally up-regulating PRLR-L. These accomplishments would accommodate account in preventing the development of OIH in women afterwards impairing the ameliorative accomplishments of opioids and acceptable additionally in appraisal of added sensitized states including, for example, stress-related affliction conditions. A added ameliorative access ability accommodate antibodies targeting PRL or the PRLR such as BAY 1158061, which do not crave axial afraid arrangement assimilation (62). In vivo gene therapies are added accepted in anesthetic and could be acclimated to adapt the complete PRLR or to access the announcement of PRLR-L (63–65). Our studies acknowledge actionable mechanisms for therapies, acceptance bigger appraisal of affliction in women. The blockage of OIH would additionally be admired in abbreviating the abrogating outcomes of opioids that accept resulted in the accepted opioid catching in the United States.

Our abstraction ambition was to analyze the mechanisms mediating beastly dimorphism in affliction manual and processing at the akin of nociceptors and to accommodate abeyant ameliorative strategies for affliction ascendancy in women. We accepted that PRLR-L in DRGs protects adjoin affliction answer by PRLR-S signaling in women, admitting down-regulation of PRLR-L mediates OIH and added injury-free affliction altitude including cephalalgia but not trauma-induced neuropathic affliction in a female-selective manner. Therefore, overexpression of PRLR-L, abatement of complete PRLR in DRGs, or blurred of circulating PRL concentrations by FDA-approved dopamine D2 agonist ability accommodate account in preventing OIH, migraine, and maybe added PRLR-S–mediated affliction altitude in women. The appraisal altar were C57BL/6 and transgenic mice as able-bodied as able DRG beef from mice. Calculation of sample admeasurement was bent on the base of antecedent abstracts appliance g-power statistics, advertence that 6 to 10 animals per accumulation are appropriate for α < 0.05. The exact n numbers acclimated in anniversary abstraction are adumbrated in the corresponding amount legends. For behavioral experiments, abstracts from animals that died or had astringent bloom problems in the average of the abstracts were afar (<5%). Abstracts were completed in assorted time periods in both macho and changeable mice, ensuring that archetype was observed. Western blotting, MEA, and immunohistochemical imaging abstracts were reproduced in assorted mice. Animals were assigned about to beginning and ascendancy groups. The experimenters were addled for beastly allocation and behavioral testing until all abstracts accumulating was complete. In the case of abstracts with afresh biologic administration, tissue collection, achievement of Western blotting and MEA experiments, and abstracts analysis, the experimenter was not blinded.

Data are bidding as agency ± SEM. Anniversary abstracts point represents an alone score. Statistical analyses were performed appliance GraphPad Prism 8 (GraphPad, La Jolla, CA). Differences amid beggarly ethics of two groups were evaluated by two-tailed Mann-Whitney test, two-tailed Student’s t test, two-tailed Wilcoxon test, and two-tailed commutual t test. Beggarly differences of added than two groups were analyzed appliance one-way appraisal of about-face (ANOVA) with Tukey’s assorted comparisons column hoc test. Differences in time-course abstracts were adjourned by two-way ANOVA with Sidak’s assorted allegory column hoc test. Differences were advised to be statistically cogent back P < 0.05. Exact P ethics and sample sizes are adumbrated in the corresponding amount legends.

stm.sciencemag.org/cgi/content/full/12/529/eaay7550/DC1

Materials and Methods

Fig. S1. Intrathecal pretreatment of BIBN 4096 prevents hindpaw allodynia induced by intrathecal bang of CGRP in naïve changeable mice.

Fig. S2. The PRL/PRLR arrangement is not analytical for the development of trauma-induced neuropathic affliction in changeable mice.

Fig. S3. Subcutaneous article of a distinct morphine pellet induces hindpaw and periorbital allodynia in changeable and macho mice.

Fig. S4. Alteration in the PRL/PRLR arrangement in changeable and macho mice with circadian injections of morphine.

Fig. S5. Schematic of circulating PRL/PRLR arrangement mediating nociceptor sensitization and OIH in females.

Table S1. Bounden affection of (−)- and ( )-oxymorphone.

Data book S1. Raw data.

References (66–74)

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