Ectopic endometrium-derived leptin produces estrogen-dependent chronic pain in a rat model of endometriosis

Abstract

Endometriosis pain is a very common and extremely disabling condition whose mechanism is still poorly understood. While increased levels of leptin have been reported in patients with endometriosis, their contribution to endometriosis pain has not been explored. Using a rodent model of endometriosis we provide evidence for an estrogen-dependent contribution of leptin in endometriosis-induced pain. Rats implanted with autologous uterine tissue onto the gastrocnemius muscle developed endometriosis-like lesions and local chronic pain. Compared to eutopic uterine tissue, leptin mRNA and protein were up-regulated in the endometriosis-like lesions. Intramuscular injection of recombinant leptin in naive rats produced dose-dependent local mechanical hyperalgesia and nociceptor sensitization to mechanical stimulation. Ovariectomy attenuated the mechanical hyperalgesia induced by recombinant leptin, in rats treated with vehicle compared to those treated with 17β-estradiol replacement, at 1 and 24 h after leptin injection. Finally, intralesional injections of a pegylated leptin receptor (Ob-R) antagonist or of an inhibitor of Janus kinase2, which transduces the Ob-R signal, markedly attenuated pain in the endometriosis model. Taken together these data support the hypothesis that leptin, generated in ectopic endometrial lesions produces mechanical hyperalgesia by acting on nociceptors innervating the lesion. This sensitivity to leptin is dependent on estrogen levels. Thus, interventions targeting leptin signaling, especially in combination with interventions that lower estrogen levels, might be useful for the treatment of endometriosis pain.

Keywords: BSA; D-PBS; DRG; Dulbecco’s phosphate-buffered saline; JAK2; Janus kinase2; Ob-R; RT-PCR; STAT3; bovine serum albumin; chronic pelvic pain; dorsal root ganglion; endometriosis; leptin; leptin receptor; mechanical hyperalgesia; nociceptor; ob gene; recombinant rat leptin; reverse transcriptase polymerase chain reaction; rrleptin; signal transducer and activator of transcription 3.

Figure 1

Surgical implant of ectopic uterine tissue on the gastrocnemius muscle produces persistent mechanical hyperalgesia and increased local levels of leptin transcripts. (A) Compared to baseline (Base) values, mechanical hyperalgesia was already present at day 10 post-implantation after unilateral transplantation of endometrium and remained unattenuated at day 21 after surgery. In contrast, rats submitted to a sham surgical procedure in the gastrocnemius muscle did not exhibit significant hyperalgesia at any time point tested; ***P < 0.001. (B) Quantitative one-step multiplex RT-PCR for leptin mRNA. Total RNA from the endometriosis-like lesion or uterine tissue from sham-operated control rats was compared 14 d post-surgery. After RT-PCR amplification, the basal concentration of mRNA encoding leptin (292 bp) was almost undetectable in eutopic endometrium (lane 1), whereas leptin mRNA was expressed in high levels in cystic lesions from implanted rats (lane 2). S18 rRNA was used as an endogenous standard (489 bp). (C) Western blot analysis. Protein extracts from ectopic (lane 1) and eutopic (lane 2) uterine tissue were submitted to Western blot analysis, using β-actin as an internal loading control.

Figure 2

Local injection of leptin produces persistent mechanical hyperalgesia. (A) Cumulative dose–response curve for the effect of i.m. rrleptin on mechanical nociceptive threshold following administration into the belly of the gastrocnemius muscle in the rat. Incremental doses from 1 ng to 1 μg were administered at 30 min intervals. (B) The time-effect curve for hyperalgesia induced by 1 μg of rrleptin was investigated from 60 min to 7 days (n = 6). Hyperalgesia reached a maximum 1 h. post-injection and lasted for 4 days. ***P < 0.001.

Figure 3

Leptin produces nociceptor sensitization. (A) Thirty min. after injection, rrleptin significantly increased the number of spikes in recordings obtained during the early part of the stimulation period (first 10 s, n = 20, P < 0.01), as well as total response (60 s, n = 20, P < 0.05), compared to pre-rrleptin injection values. One hour after injection, rrleptin not only increased the number of spikes in recordings obtained during the early part of the stimulation period (first 10 s, n = 20, P < 0.01), but also in its late component (last 50 s, n = 20, P < 0.05) and total response (60 s, n = 20, P < 0.05) compared to pre-injection values. This increased response at 1 h. after rrleptin injection was also evident in histograms depicting the time-course of the nociceptor response, representing recordings obtained pre and post rrleptin injection (B-D); *P < 0.05; **P < 0.01.

Figure 4

Leptin-induced hyperalgesia is estrogen-dependent. Ovariectomized rats were treated with either 17β-estradiol or vehicle for 3 consecutive days. One day after, they received an i.m. injection of rrleptin (1 μg/20 μl) and muscle mechanical nociceptive threshold was measured 1 or 24 h after rrleptin injection. Rats without estrogen replacement exhibited a marked attenuation of the nociceptive response induced by rrleptin; ** P < 0.001.

Figure 5

Intralesional leptin receptor antagonist inhibits mechanical hyperalgesia in rats submitted to a model of endometriosis pain. Two weeks after surgical implant of autologous uterine tissue, rats displayed a marked mechanical hyperalgesia (EMS, endometriosis pain model). (A) Single intralesional injection of leptin receptor pegylated antagonist (3 μg/20 μl), but not vehicle (Veh), produced an attenuation of mechanical hyperalgesia at 1 h after injection which reached a maximal effect 24 h. after injection; (B) Repeated intralesional injections of leptin receptor pegylated antagonist (3 μg/20 μl*3) produced an inhibition of mechanical hyperalgesia lasting at least 3 days after last injection. (C) Administration of leptin receptor pegylated antagonist (3 μg/20 μl) into the contralateral gastrocnemius muscle was devoid of effect. *P < 0.05; ***P < 0.001.

Figure 6

Inhibition of leptin receptor signalling attenuates mechanical hyperalgesia in rats submitted to a model of endometriosis pain. Two weeks after surgical implant of autologous uterine tissue, rats displayed a marked mechanical hyperalgesia (EMS, endometriosis pain model). In these rats, a single local injection of the selective inhibitor of JAK2 (AG490), but not vehicle (Veh), produced an attenuation of mechanical hyperalgesia between 1 to 24 h. after injection; ***P < 0.001.