Preclinical evaluation of pain in endometriosis

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1 receptor antagonism reduces peripheral and central hyperalgesia in a preclinical mouse model of endometriosis Erin Greaves, Andrew W Horne, Helen Jerina, arta ikolajczak, Lisa Hilferty, Rory itchell, Sue Fleetwood-Walker, Philippa TK Saunders

2 Supplementary Information Supplementary Tables Supplementary Table ; Information about Lesions Recovered from the ouse odel Sample size Recovery rate 9% Number of lesions (mean±se).9±. Supplementary Table : Primer sequences. Gene Forward Reverse UPL Probe 5 -cagaggagacggaccacct- 5 -ccatgtaggcaaagattgtgaa- 5 -cctaaccccaccctacaggt- 5 agaaggacgcgttgactcc- 77 COX- 5 -cctctttccaggagctcaca- 5 -tcgatgtcaccgtacagctc- 6 COX- 5 -gatgctcttccgagctgtg- 5 -ggattggaacagcaaggattt- 5 TRPV 5 -caacaagaaggggcttacacc- 5 -tctggagaatgtaggccaagac- SCNA 5 -ttcataatgtgtggcaactgg- 5 -ttattgcacgtggaaccatc- 9 Supplementary Table : Antibodies. Antibody Raised in Application Supplier Dilution Rabbit Immunohistochemistry Cayman; 75 : Immunohistochemistry Cayman;775 :5 COX- Immunohistochemistry Bio-Vision; 6- :5 COX- (H-6) Rabbit Immunohistochemistry Santa Cruz Biotech; :5 sc-795 TRPVI Guinea pig Dual immunofluorescence Abcam; ab95 : Peripherin Chicken Dual immunofluorescence Abcam; ab97 :5 NF- Chicken Dual immunofluorescence illipore; SS9 : 5 Rabbit Dual Immunofluorescence Alomone; APR-6 :

3 COX- Rabbit Western blot Santa-Cruz Biotech; : sc-795 GAPDH ouse Western blot illipore; AB 7 : Supplementary Table. It has been reported that the expression in mouse uterus of standard housekeeping genes such as GAPDH can be altered by E treatment (Schroder et al., 9). However, in the CNS regions assessed here, the OVX + E and endometriosis model groups showed no discernible changes in GAPDH expression compared to naïve mice. For equal gel loading volumes of lysates produced at a fixed ratio of tissue wet weight per ml, we found densitometric measurements of GAPDH expression in the OVX + E group to be.9 ±.9%,. ± 6.% and 7. ± 6.% of naïve for spinal cord, thalamus and anterior cingulate cortex respectively (means ± SE, n = 5-6). Corresponding values for the endometriosis model group were 96.6 ± 6.%, 96.5 ± 5.% and 95. ± 6.5%. Analysis by One-Way ANOVA with Dunnett s post-hoc test indicated no statistically significant differences in any case, validating our use of GAPDH here as a comparator housekeeping gene.

4 Supplementary Figures A 5 Number of lesions vs withdrawal threshold-abdomen B 5 Number of lesions vs withdrawal threshold-hindpaw Number of lesions Number of lesions Withdrawal threshold (g) Paw withdrawal threshold (g) Fig.S Endometriosis mice show no correlation between mechanical hyperalgesia and number of lesions. Correlation between withdrawal threshold (g) and number of lesions when von Frey filaments were applied to a) the abdomen and b) the hindpaw. N= mice.

5 Preclinical evaluation of pain in endometriosis a i 5 ii iii iv GE S S S b i. ii iii iv GE.5. S S S.5. c i 5 COX- ii S iii iv S S GE d i COX- ii.6 iii iv S. S S * GE.. e i * 6 f PGE pg/ml Naive OVX+E Endo g

6 Fig.S The PGE signaling pathway is over-expressed in endometriosis lesions. mrna concentrations and protein expression of (a), (b), (c) COX-, (d) COX- in peritoneum from naïve mice (; ii), peritoneum from mice with endometriosis (; iii) and endometriosis lesions (; iv). (i) Graphs indicate QPCR data where values are normalized to expression in samples of cycling uterus., n=9,, n=6 and, n=8. : Relative quantification. (ii-iii) Representative images of single antigen immunodetection taken from, and. Insets are negative controls (omission of primary antibody on sections of cycling uterus). Scale bars = 5 or µ. (e) Peritoneal fluid concentrations of PGE. PGE concentrations (pg/ml) were measured using ISA in the peritoneal fluid of mice with endometriosis (n=) compared to naïve (n=8), and OVX+E mice (n=7). (f-g) mrna concentrations of (f) and (g) were also analyzed in lesions and peritoneum, there was no significant difference in expression levels and therefore these particular receptors were not investigated further. Statistical analysis was performed using a one-way ANOVA and Newman-Keuls post comparison test. *p<.5, p<., p<.. 6

7 Preclinical evaluation of pain in endometriosis a b..5 c COX-.5 OVX+E Endo OVX+E. OVX+E Endometriosis Endo d TRPV % TRPV + NF- co-localizaton * OVX+E Endo erge NF- OVX+E Endometriosis f % + TRPV co-localization 8 6 OVX+E Endo erge TRPV e Fig.S Gene expression in DRGs of endometriosis mice. (a-b) QPCR analysis of the prostaglandin E receptor (a), and (b) COX- in L5-L6 DRGs from mice with endometriosis (n=9) compared to 7

8 naïve (n=7) and OVX+E control mice (n=6). : Relative quantification. Values were normalized to a single naïve DRG sample given the arbitrary value of one. eans were not significantly different. (cd) Dual label immunofluorescence was carried out to identify TRPV expression (red) in L5-6 DRG cells co-expressing neurofilament- (NF-; green). (c) Shows typical confocal images for TRPV and NF- from naïve, OVX+E and endometriosis mice (field of view, 6x6µm). Total cells counted were, 68, and, accumulated from three different naïve, OVX+E -treated and endometriosis mice in each case. (d) Shows a bar chart that summarizes % expression of TRPV in NF- -positive cells and indicates that the number of TRPV+NF-+ small DRG cells is significantly increased in mice with endometriosis. Statistical analysis was performed using a one-way ANOVA and Tukey s post-hoc test. *p<.5. (e-f) Dual label immunofluorescence was carried out to identify expression (red) in L5-6 DRG cells co-expressing TRPV (cyan). (e) Showes typical confocal images for and TRPV from naïve, OVX+E and endometriosis mice (field of view, 6x6µ). Total cells counted were 9, 58, 6, accumulated from three different naïve, OVX+E -treated and endometriosis mice in each case. (f) Shows a bar chart that summarises % expression of in TRPV- positive cells and indicates that the number of +, TRPV+ small DRG cells is significantly increased in endometriosis. Statistical analysis was performed using a one-way ANOVA and Tukey s post-hoc test. p<.. 8

9 a echanical withdrawal threshold (g) c echanical withdrawal threshold (g) e echanical withdrawal threshold (g) g echanical withdrawal threshold (g) TRPV antagonist - abdomen JNJ * 6 8 antagonist - abdomen L-698 * * 6 8 * +++ antagonist - abdomen TG antagonist - abdomen PF Naive + drug Endo + drug Endo + vehicle b d f h 5 TRPV antagonist - hind-paw JNJ antagonist - hind-paw L antagonist - hind-paw TG antagonist - hind-paw PF Fig.S Time-course of effects on local and secondary mechanical hyperalgesia of i.p injection of potential therapeutics in a mouse model of endometriosis. Graphs depict withdrawal thresholds for von Frey filaments applied to the rostral regions of the abdomen or hindpaws of endometriosis mice 9

10 compared to naïve controls. OVX+E mice were not tested because Figure indicated that their responses were unaltered from naïve control mice. (a) Shows the time-course of effects of the TRPV inhibitor JNJ 7 ( mg/kg ip) on abdominal and (b) hindpaw mechanical withdrawal responses in endometriosis mice (blue and pink lines) or naïve controls (purple lines). In both abdomen and paw tests, withdrawal thresholds were significantly lower in endometriosis mice than in naïve mice (p<. and p<.). These differences were modestly but significantly attenuated with JNJ 7 (p<.and p<.5), which had no discernible effect in naïve controls. (c) Shows the time-course effects of the antagonist TG6-- (mg/kg, ip), on abdominal and (d) paw mechanical withdrawal threshold in endometriosis mice compared to naïve controls. In both abdomen and paw tests, withdrawal thresholds were significantly lower in endometriosis mice compared to naïve controls (p<.5 and p<.). These differences were substantially and significantly attenuated by TG6-- (p<. and p<.). TG6-- had no discernible effect in naïve controls. The highly selective antagonist PF- 898 (mg/kg, ip) substantially and significantly reversed (e) abdominal and (f) hindpaw mechanical hyperalgesia (p<.). There was no discernible effect of PF-898 in naïve mice. The selective antagonist L-698 had only a very small but statistically significant effect on (g) abdominal mechanical hyperalgesia and no discernible effect on (h) paw mechanical hyperalgesia (n=5 in all cases). Statistical analysis was performed using a Two-Way ANOVA with a Dunnett s multiple comparison test. *p<.5, p<. and p<. compared to naïve mice + pharmacological agent. +p<.5, ++p<. and +++p<. compared to pre-administration baseline.