The effects of cycling levels of 17beta-estradiol and progesterone on the magnitude of temporomandibular joint-induced nociception

Abstract

A greater incidence of temporomandibular joint (TMJ) pain is reported in females, suggesting that gonadal hormones may play a role in this condition. However, the exact roles of 17beta-estradiol (E2) and progesterone (P4) in TMJ pain are not completely known. Two experiments were performed to determine the separate roles of E2 and P4 in TMJ nociception at various stages of the estrous cycle. Ovariectomized (OVX) rats were cycled with physiological concentrations of E2 or P4. The E2-cycled rats then received bilateral TMJ injections of saline (SAL) or complete Freund's adjuvant (CFA) on the morning of diestrus-2 (low E2 condition) or proestrus (high E2 condition). As a control, OVX rats (no ovarian E2 and no replacement) were injected with SAL or CFA. The TMJ nociception was measured using a validated novel method in which an increase in meal duration directly correlated to the intensity of deep TMJ nociception. In the E2 experiment, CFA injection, but not SAL, increased TMJ nociception in the OVX group, but the effect was less pronounced in diestrus-2 and even less in proestrus. In the P4 experiment, the rats receiving TMJ CFA in diestrus-2 (end of minor P4 surge) did not show an increase in TMJ nociception, whereas the rats injected in proestrus (major P4 surge), estrus (low P4), and metestrus (low P4) had similar increases in TMJ nociception. The hormones' concentration did not affect TMJ IL-1beta, IL-6, C-C motif ligand 20, or C-X-C motif ligand 2 or the trigeminal ganglia calcitonin gene-related peptide. The high physiological concentrations of E2 observed at proestrus and the low P4 concentrations observed at diestrus-2 attenuated or eliminated CFA-induced TMJ nociception. The results suggest that the cyclic estrous cycle concentrations of E2 and P4 can influence CFA-induced TMJ nociception in the rat.

Figure 1

A, Time line for the 17β-estradiol experiment protocol. The experiment included 17β-estradiol replacement in ovariectomized (OVX) rats, TMJ injections, measurements of meal duration, and killing of animals for collection of tissues. Injections of 17β-estradiol on d 5 (estrous cycle 1), 10 (estrous cycle 2), and 15 (estrous cycle 3) resulted in a proestrus surge during each cycle. B, The progesterone experiment time line includes progesterone replacement in OVX rats, TMJ injections, measurements of meal duration, and killing of animals for collection of tissues. Injections of low doses of progesterone on diestrus-1 d 2 (estrous cycle 1), 7 (estrous cycle 2), and 12 (estrous cycle 3) caused the low diestrus-1/diestrus-2 surge for each cycle. Injections of progesterone on d 4 (estrous cycle 1), 9 (estrous cycle 2), and 14 (estrous cycle 3) caused the proestrus surge for each cycle.

Figure 2

The concentration (nanograms hormone per milliliter of blood plasma) of 17β-estradiol (A) and progesterone (B) was measured during a 5-d estrous cycle of intact rats [□; redrawn with permission from Butcher et al. (31)]. The concentration of 17β-estradiol (A) and progesterone (B) in the plasma of ovariectomized (○) rats was measured after administering replacement of 17β-estradiol (A) and progesterone (B). M, Midnight. Six rats were in each experimental group. C, Daily body weight change in female rats. Groups include ovariectomized (OVX) females given vehicle (sesame seed oil) injections sc (OVX+vehicle), intact female rats (intact), OVX females cycled with 17β-estradiol (OVX+17β-estradiol), and OVX females cycled with progesterone (OVX+progesterone). Body weights were calculated by subtracting the body weight on the day before the beginning of the second cycle from the body weight during each phase of the second estrous cycle. *, P < 0.05; **, P < 0.01 (two-way ANOVA) when comparing the mean body weight values of OVX+17β-estradiol and intact rats vs. the OVX+vehicle-treated rats. Representative vaginal smears from intact cycling rats (left side, D, F, H, and J) and OVX rats (right side, E, G, I, and K) given an artificial estrous cycle as described in Fig. 1A. Panels D and E are from rats in diestrus, panels F and G are from rats in proestrus, panels H and I are from rats in estrus, and panel J and K are from rats in metestrus. Cell were stained with thiazine blue; bar on all images, 20 μm.

Figure 3

17β-Estradiol experiment 24-h meal duration measurements. A, Graph showing the meal duration of rats that were ovariectomized (OVX) or in diestrus-2 or proestrus. Data are from estrous cycle before cycle when they were given TMJ injection. B, Meal duration from rats during different phases of the third estrous cycle after TMJ injection of saline or CFA. Meal duration measurements were completed by placing rats in sound-attenuated chambers equipped with photobeam computer-activated 45-mg rodent chow pellet feeders. A meal was defined as when the rat does not take a single pellet for 10 min. The duration of the meals for each rat during a 24-h period was calculated, and the values for each treatment group were pooled and plotted as a mean ± sem. A three-way ANOVA [estrous stage, CFA, or saline and time (before and after TMJ injection)] was performed and significant data further analyzed using Tukey’s post hoc test. CFA significantly lengthened meal duration in all groups. **, P < 0.01. The OVX CFA had a significantly longer meal duration than the diestrus-2 (*, P < 0.05) and proestrus groups (**, P < 0.01). The meal duration of the diestrus-2 group was significantly longer than the proestrus group (**, P < 0.01). Mean ± sem.

Figure 4

Molecules analyzed in the 17β-estradiol experiment. Ovariectomized (OVX) rats and rats having proestrus levels of 17β-estradiol had their TMJ tissue (i.e. retrodiscal, disk, and synovium) removed 24 h after injecting saline or CFA into the TMJ. The amount of cytokines IL-1, IL-1β (A), IL-6, IL-6 (B), and CCL20 (C), and CXCL2 (D) were quantitated by ELISA. The amount of CGRP (E) in the trigeminal ganglia was measured 24 h after saline or CFA injection by RIA. Values for the cytokine or chemokines or CGRP concentration was calculated per milligram of total protein in the tissue. Data were compared by two-way ANOVA and significant data analyzed by Tukey’s post hoc test. *, P < 0.05; **, P < 0.01. Mean ± sem.

Figure 5

Progesterone experiment 24-h meal duration measurements. A, Graph shows the meal duration of rats at different phases of the second estrous cycle. Data from estrous cycle before cycle when they were given TMJ injection. B, Meal duration from rats during different phases of the third estrous cycle after TMJ injection of saline or complete Freund’s adjuvant (CFA). A three-way ANOVA [estrous stage, CFA or saline and time (before and after TMJ injection)] was performed and significant data further analyzed using Tukey’s post hoc test. CFA significantly lengthened meal duration in all groups, except the diestrus-2 group. **, P < 0.01. The TMJ CFA-injected diestrus-2 group’s meal duration was not different from the diestrus-2 TMJ saline-injected group, but it was significantly shorter than the TMJ CFA-injected proestus, estrus, and metestrus groups. **, P < 0.01. Mean ± sem.

Figure 6

Molecules analyzed in the progesterone experiment. Rats having diestrus-2 or estrus levels of progesterone had their TMJ tissue (i.e. retrodiscal, disk, and synovium) removed 24 h after injecting the TMJ with saline or CFA. The amount of IL-1, IL-1β (A), IL-6, IL-6 (B), CCL20 (C), and CXCL2 (D) were quantitated by ELISA. The amount of CGRP (E) in the trigeminal ganglia was measured 24 h after saline or CFA TMJ injections by RIA. Values for the cytokine or chemokines or CGRP concentration was calculated per milligram of total protein in the tissue. Data were compared by two-way ANOVA and significant data analyzed by Tukey’s post hoc test. *, P < 0.05; **, P < 0.01. Mean ± sem.