The Effects of Cycling Levels of 17β-Estradiol and Progesterone on the Magnitude of Temporomandibular Joint-Induced Nociception

NEUROENDOCRINOLOGY The Effects of Cycling Levels of 17␤-Estradiol and Progesterone on the Magnitude of Temporomandibular Joint-Induced Nociception P. R. Kramer and L. L. Bellinger Department of Biomedical Sciences, Texas A&M Health Science Center, Baylor College of Dentistry, Dallas, Texas 75246 ysfunctional and disabling conditions of the temporomandibular joint (TMJ) afflict an estimated 6 –15% of the population in North America (1). The question of whether clear sex differences exist in the signs and symptoms of the disorders of the TMJ (TMD) remains unresolved. Nevertheless, epidemiological studies have indicated that women do seek treatment for TMD much more often than men, to the extent that they comprise more than three fourths of the clinical cases (2). Moreover, women appear to exhibit more severe symptoms of TMD (3, 4). Although the biological basis for this possible sex-based disparity is unclear, the time course of symptoms associated with TMD is noteworthy in females. The peak incidence of TMD occurs during the child-bearing years (5), varies in intensity over the men- D strual cycle (6, 7), and is marked by the attenuation of symptoms in postmenopausal women (8, 9). However, the attenuation of symptoms in postmenopausal women is reversed when they take estrogens supplements (10, 11). In acute animal studies, pharmacological doses and/or the nonphysiological application of 17␤-estradiol have been reported to cause an increased or decreased sensitivity to noxious sc stimuli over the rat TMJ region (12–15). Estrogens have also been shown to decrease sensitivity to noxious sc stimuli over the human female TMJ region (16). The effects of progesterone on nociception are also complex; as with estrogens (3, 17), testing has been performed using a variety of methods. Limited testing of the effect of progesterone ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-1707 Received December 5, 2008. Accepted April 2, 2009. First Published Online April 9, 2009 Abbreviations: CCL20, C-C motif ligand 20; CFA, complete Freund’s adjuvant; CGRP, calcitonin gene-related peptide; CXCL2, C-X-C motif ligand 2; TMD, disorders of the TMJ; TMJ, temporomandibular joint. 3680 endo.endojournals.org Endocrinology, August 2009, 150(8):3680 –3689 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 17␤-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-1␤, 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. (Endocrinology 150: 3680 –3689, 2009) Endocrinology, August 2009, 150(8):3680 –3689 3681 duration assay to study TMJ nociception, which can detect changes in TMJ nociception over the course of days in an undisturbed rat. To determine the mechanism(s) by which 17␤estradiol and progesterone might influence TMJ nociception, we measured the changes in TMJ inflammatory IL-1 and -6, TMJ inflammatory chemokines C-C motif ligand 20 (CCL20) and C-X-C motif ligand 2 (CXCL2), TMJ calcitonin gene-related peptide (CGRP) and trigeminal ganglia CGRP. Materials and Methods Female (250 g) Sprague Dawley rats were ovariectomized (Harlan Industries, Houston, TX). On arrival, the rats were kept on a 14-h light, 10-h dark cycle with lights on at 0600 h. The animals were housed individually in our computerized feeding modules and given food and water ad libitum. They were acclimated to the surroundings for 2 d before receiving hormone replacement. Artificial estrous cycle using 17␤-estradiol To produce physiological, cycling levels of 17␤-estradiol, the ovariectomized rat required a basal level of hormone and a proestrus surge of hormone (Fig. 1A). For baseline hormone replacement, the rats were anesthetized with ketamine (52 mg/kg) and xylazine (5.4 mg/kg). Using a sterile technique, the rats were implanted with 28-d Alzet miniosmotic pumps that were primed according to the manufactures directions (Durect Corp., Cupertino, CA) and on implantation were immediately dispensing 6 ␮l/d of the vehicle polyethylene glycol (no 17␤-estradiol replacement control group) or 750 ng per 6 ␮l/d 17␤-estradiol benzoate (Sigma, St. Louis, MO) (23, 30). This dose of 17␤-estradiol benzoate produces plasma 17␤-estradiol concentrations of approximately 10 pg/ml (30), a concentration similar to those reported in diestrus-1, diestrus-2, estrus, and metestrus (31–33). In addition, the rats receiving 17␤-estradiol replacement were injected sc at 0800 h every 5 d with 2.5 ␮g 17␤-estradiol benzoate in 0.1 ml sesame oil; this amount approximates the proestrus surge (60 –70 pg/ml) in plasma 17␤-estradiol (31, 32). The control rats received oil injections. The dose and timing of the proestrus 17␤-estradiol benzoate injections were determined in a series of pilot studies. First, plasma 17␤estradiol measurements were made throughout proestrus (five to six rats per group) after various doses of 17␤-estradiol. The plasma 17␤-estradiol levels were then determined by RIA (Diagnostic Systems Laboratories, Webster, TX) and compared with the levels in intact rats (31, 33). These measurements determined the dose strategy. Second, because ovariectomized rats show excessive body weight gain due to the lack of 17␤-estradiol and that ovariectomized rats receiving 17␤-estradiol do not (34, 35), body weights of the ovariectom (...truncated)