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. 2017 Mar 10:7:44169.
doi: 10.1038/srep44169.

EP2 receptor antagonism reduces peripheral and central hyperalgesia in a preclinical mouse model of endometriosis

Erin Greaves  1 , Andrew W Horne  1 , Helen Jerina  2 , Marta Mikolajczak  2 , Lisa Hilferty  1 , Rory Mitchell  2 , Sue M Fleetwood-Walker  2 , Philippa T K Saunders  3
Affiliations

EP2 receptor antagonism reduces peripheral and central hyperalgesia in a preclinical mouse model of endometriosis

Erin Greaves et al. Sci Rep. .

Abstract

Endometriosis is an incurable gynecological disorder characterized by debilitating pain and the establishment of innervated endometriosis lesions outside the uterus. In a preclinical mouse model of endometriosis we demonstrated overexpression of the PGE2-signaling pathway (including COX-2, EP2, EP4) in endometriosis lesions, dorsal root ganglia (DRG), spinal cord, thalamus and forebrain. TRPV1, a PGE2-regulated channel in nociceptive neurons was also increased in the DRG. These findings support the concept that an amplification process occurs along the pain neuroaxis in endometriosis. We then tested TRPV1, EP2, and EP4 receptor antagonists: The EP2 antagonist was the most efficient analgesic, reducing primary hyperalgesia by 80% and secondary hyperalgesia by 40%. In this study we demonstrate reversible peripheral and central hyperalgesia in mice with induced endometriosis.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Behavior testing in control and endometriosis mice.
Endometriosis mice, together with ovariectomised, estradiol-treated sham recipients, with or without i.p injection of PBS, and naïve controls were scored for general behaviors indicative of discomfort that might be associated with pelvic pain. (a) Shows abdominally directed licking (average number of grooming events recorded by two observers over two 5 min periods) was significantly increased in endometriosis mice (n = 5) compared to estradiol-treated (OVX + E2; n = 3, OVX + E2 + PBS; n = 6) and naïve controls (n = 7). (b) Shows that exploratory activity (average number of open-field tunnel entries recorded by two observers was significantly reduced in endometriosis mice (n = 9) compared to estradiol-treated (OVX + E2; n = 6, OVX + E2 + PBS; n = 6) and naïve controls (n = 11). Mechanical withdrawal threshold, shown by von Frey filament testing, was also measured on the lower abdomen and plantar hind-paw of endometriosis mice (n = 6), OVX + E2, OVX + E2 + PBS (n = 6) and naïve controls (n = 7). For quantitative sensory testing, von Frey filaments were applied to caudal regions of the abdomen; (c and d) show that withdrawal thresholds (g = grams) for both abdomen and paw testing were significantly decreased in endometriosis mice compared to the other groups. Statistical analysis was performed using a one-way ANOVA and Newman-Keuls post-hoc test (a and b) or a Kruskall-Wallis test and Dunn’s multiple comparison test. *p < 0.05. **p < 0.01, ***p < 0.001.
Figure 2
Figure 2. Molecular changes in the nervous system of mice with endometriosis.
Expression of the PGE2 pathway and nociceptive ion channels is elevated in the DRGs, spinal cord and brain of mice with endometriosis. (a–d) QPCR analysis of the prostaglandin E receptor 2(a) EP2, (b) COX-1 and nociceptive ion channels (c) SCN11A and (d) TRPV1 in L5-L6 DRGs from mice with endometriosis (n = 9) compared to naïve (n = 7) and OVX + E2 control mice (n = 6). RQ: Relative quantification. Values were normalized to a single naïve DRG sample given the arbitrary value of one. (e,f) Dual label immunofluorescence was carried out to identify TRPV1 expression (red) in L5-6 DRG cells co-expressing peripherin (green). (e) Shows typical confocal images for TRPV1 and peripherin from naïve, OVX + E2-treated sham-recipient and endometriosis mice (field of view, 160 × 160 μm). Total cells counted were 330, 320, and 543, accumulated from three different naïve, OVX + E2-treated and endometriosis mice in each case. (f) Shows a bar chart that summarizes % expression of TRPV1 in peripherin-positive cells and indicates that the number of TRPV1 + peripherin + small DRG cells is significantly increased in mice with endometriosis. (g–i) Images (i) show representative examples of COX-2 (top panel; 80 kDa) and GAPDH (bottom panel; 36 kDa) expression as analyzed using Western blot in (g) spinal cord, (h) thalamus, (i) and anterior cingulate cortex of endometriosis mice, OVX + E2-treated sham-recipients and naïve controls, n = 5-6 for all groups. (ii) Graphs showing COX-2: GAPDH densitometric ratio derived from quantitative densitometry of films. Both images and bar charts indicate marked elevation of COX-2 expression in each region. No changes were observed in OVX + E2-treated and naïve controls. Statistical analysis was performed using a one-way ANOVA and Tukey’s post comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3. Pre-clinical testing of potential therapeutics in a mouse model of endometriosis.
Graphs depict 50% mechanical withdrawal thresholds for von Frey filaments (g = grams) applied to abdomen or hindpaw of mice with endometriosis or naïve controls, n = 5 all groups. The time-point showing maximal reversal of pain is shown for each antagonist. (a) Effects of the TRPV1 inhibitor JNJ 17203212 (30 mg/kg ip), 30 mins post-injection on abdominal withdrawal responses in mice with endometriosis compared to naïve mice. (b) Shows corresponding results from hindpaw. Concurrently measured naïve values were 0.55 ± 0.17 g and 11.25 ± 1.44 g respectively for abdomen and hindpaw. In both tests, withdrawal thresholds were significantly lower in mice with endometriosis (Endo + Vehicle) than in naïve controls or in naïve animals treated with the drug (Naïve + JNJ; p < 0.01 and p < 0.001). This difference was modestly but not significantly attenuated by JNJ 17203212, which had no discernible effect on the responses of naïve animals. (c) Effects of the selective EP4 antagonist L-161982 (10 mg/kg ip) 30 mins post i.p injection on abdominal and (d) hindpaw mechanical withdrawal thresholds. Naïve values were 0.39 ± 0.07 g and 9.43 ± 1.43 g. In both tests, thresholds were significantly lower in mice with endometriosis than in naïve animals treated with the drug (p < 0.001). L-161982 had no discernible effect on hypersensitivity in mice with endometriosis or in naïve animals. (e) Effect of the EP2 antagonist TG6-10-1 (10 mg/kg, ip), on abdominal and (f) hindpaw mechanical withdrawal thresholds at 45 mins post i.p injection. Naïve values were 0.31 ± 0.11 g and 9.69 ± 1.62 g. In both tests, thresholds were significantly lower in mice with endometriosis compared to naïve controls or in naïve animals treated with the drug (p < 0.01 and p < 0.001). This difference was significantly reversed by TG6-10-1 in abdominal tests (p < 0.05). TG6-10-1 also induced a modest but not statistically significant pain reversal in paw tests. The highly selective EP2 antagonist PF-04418948 (10 mg/kg, i.p) significantly attenuated both (g) abdominal and (h) hindpaw mechanical hypersensitivity in mice with endometriosis (p < 0.001) 45 mins post injection. There was no discernible effect of PF-04418948 in naïve animals. Naïve values were 0.48 ± 0.05 g and 8.77 ± 0.51 g. Statistical analysis was performed using a one-way ANOVA and Tukey’s post comparison test. *p < 0.05, **p < 0.001, ***p < 0.001.
Figure 4
Figure 4. Oral administration of the selective EP2 antagonist PF-04418948.
To test the efficacy of oral administration of PF-04418948, the drug was administered at a dose of 10 mg/kg by oral gavage and testing was initiated after 30 min. The graphs show the time-course of effects of PF-04418948 on mechanical withdrawal responses in mice with endometriosis (blue and pink lines) or naïve controls (purple lines). In both (a) the abdomen and (b) the hindpaw, withdrawal thresholds were significantly lower in endometriosis mice than in naïve controls (p < 0.001). The pre-drug withdrawal thresholds in the naïve, drug-treated animals, were not discernibly different from those in naïve, untreated animals (0.55 ± 0.06 g for abdomen and 9.00 ± 0.67 g for hindpaw). In each case the hypersensitivity due to endometriosis was substantially and significantly attenuated by PF-04418948 (p < 0.001), n = 5 all groups. Statistical analysis was performed using a Two-Way ANOVA and Dunnett’s multiple comparison test. *p < 0.05, **p < 0.001 and ***p < 0.001 compared to naïve mice + pharmacological agent. +p < 0.05, ++p < 0.01 and +++p < 0.001 compared to pre-administration baseline.
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References

    1. Rogers P. A. et al.. Priorities for endometriosis research: recommendations from an international consensus workshop. Reprod Sci 16, 335–346 (2009). - PMC - PubMed
    1. Dunselman G. A. et al.. ESHRE guideline: management of women with endometriosis. Hum Reprod 29, 400–412 (2014). - PubMed
    1. Giudice L. C. & Kao L. C. Endometriosis. Lancet 364, 1789–1799 (2004). - PubMed
    1. Stratton P. & Berkley K. J. Chronic pelvic pain and endometriosis: translational evidence of the relationship and implications. Hum Reprod Update 17, 327–346 (2011). - PMC - PubMed
    1. Holzer P. Neurogenic vasodilatation and plasma leakage in the skin. Gen Pharmacol 30, 5–11 (1998). - PubMed

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