Skip to main content
. 2019 Jul 10;33(10):11210–11222. doi: 10.1096/fj.201900797R

Figure 2.

Figure 2

Macrophages play a key role in endometriosis-associated hyperalgesia. A) Schematic of endometriosis model indicating timing of behavioral tests and liposome–saline administration. B) Representative flow plots showing peritoneal lavage of saline control (left panel) and liposomal clodronate depleted (right panel). Myeloid cells were separated into 4 populations using F4/80 and Ly6C; these were F4/80hi, Ly6C− (LpMs), F4/80hi, Ly6C+ (MDMs), F4/80lo, Ly6Clo (SpMs), and Ly6C+, F4/80− (monocytes). C–F) Quantification of flow cytometry results normalized to 123count eBeads to allow resolution of absolute numbers (naive mice, n = 10; sham-treated mice, n = 9; Endo, n = 14; Endo + Lipo, n = 17). C, D) Clodronate administration caused depletion of LpMs (P < 0.001) (C) and MDMs (P < 0.05) (D). E) SpMs were not significantly altered. F) Monocytes were significantly increased in depleted endometriosis mice compared with naive and sham-treated animals (P < 0.05). G) Macrophages in the lesions of mice were visualized using F4/80 (red) immunofluorescence. Scale bar, 50 μM. Inset on right panel is negative control (negative primary antibody). H) Macrophage numbers [3 × random fields of view (FOVs) per lesion quantified] were reduced (P < 0.01) in the lesions of mice that received liposomal clodronate (n = 9 mice) compared with saline (n = 13 mice). I) In C57BL/6 mice with endometriosis (n = 14), abdominally directed grooming was elevated compared with naive (n = 10; P < 0.05) and sham-treated animals (n = 9; P < 0.01). Macrophage depletion (n = 17) decreased grooming levels (P < 0.05). J) Exploratory behavior was reduced in mice with endometriosis (P < 0.01). Macrophage depletion did not rescue endometriosis-associated changes in activity. K) Macrophage depletion increased abdominal (P < 0.01) retraction thresholds. L) Paw withdrawal thresholds were lower in endometriosis mice compared with naive and sham-treated animals (P < 0.001), and macrophage depletion attenuated hind-paw withdrawal thresholds. A single time point (d 26) that exhibited maximal changes in sensory behavior is shown. A Mann-Whitney U test, (H), 1-way ANOVA, and Tukey’s multiple comparison test (I, J) or a Kruskal-Wallis nonparametric test with Dunn’s multiple comparison test were performed (K, L). Lin, lineage; Lipo, liposome; OVX, ovariectomy. *P < 0.05, **P < 0.01, ***P < 0.001.