Measuring persistent temporomandibular joint nociception in rats and two mice strains

Abstract

Temporomandibular joint (TMJ) pain has been reported to last for prolonged periods in humans. In rodents a variety of methods have been used to measure TMJ nociception, but for most of these methods the period of measurement has been minutes to a couple of hours. In addition, most measurement protocols required restraint or training of the animal. Previous studies from our laboratory demonstrated that feeding behavior, particularly meal duration, was an indicator of TMJ nociception in unrestrained and untrained male and female Sprague-Dawley rats for up to two days. In this study, we first found that injection of complete Freund's adjuvant (CFA) into the TMJ of rats significantly lengthened meal duration for 19 days and also decreased meal frequency for 42 days. Interestingly, the meal duration varied significantly from day to day within the 19 day period. TMJ interleukin-1 beta (IL-1 beta) and calcitonin gene-related peptide (CGRP) were significantly elevated in the TMJ tissues of CFA-injected animals and the level of these markers was attenuated as the meal duration decreased with time. Control animals injected with saline into the TMJ or CFA into the knee did not show a significant lengthening in meal duration but did show a decrease in meal frequency. In a second study, DBA/1LacJ mice given TMJ CFA injections showed a significantly lengthened meal duration on four of the seven days measured using end-of-the meal definition of 5 or 10 min. No other meal pattern changed significantly. Two days post-CFA injection, the DBA/1LacJ mice showed significantly elevated interleukin-6 (IL-6), but not elevated IL-1 beta. Seven days post-injection, both IL-6 and IL-1 beta were significantly elevated. No change in CGRP was detected. In this study C57Bl/6 mice also received TMJ CFA injections, but they did not show a lengthening in any meal pattern or significant increases in IL-1 beta, IL-6 or CGRP. Our data show, for the first time, that meal duration can be used to measure CFA-induced nociception in the TMJ over the course of several weeks in unrestrained rats and for up to seven days in the DBA/1LacJ mouse strain. In addition, C57Bl/6 mice are resistant to CFA-induced TMJ nociception at the same dose used in the DBA/1LacJ mice.

Figure 1

A) Dark phase meal duration and B) dark phase meal frequency are shown for Sprague-Dawley rats after bilateral injection of 50 μl saline (SALINE) into the temporomandibular joint (TMJ) (SALINE/TMJ, n = 13) or 250 μg of complete Freund's adjuvant (CFA) into the TMJ (CFA/TMJ, n = 14) or knee (KNEE/CFA, n = 7). Data include pre-day (0) to TMJ injection through 42 days after injection. A 10 minute end-of-the-meal definition was used in the calculations for these graphs. Values are given as the means ± SEM. SALINE/TMJ group compared to the CFA/TMJ group, a = P<0.05 and CFA/TMJ group as compared to the CFA/KNEE group, b = P<0.05. SALINE/TMJ group compared to the CFA/KNEE group, *=<0.05.

Figure 2

Meal duration measurements are shown for representative Sprague-Dawley rats over a three week period. Panels A-F show 24-hour meal duration values for six different rats over a 21 day period in the SALINE/TMJ group. Panels G-L shows 24-hour meal duration values for six different rats in the CFA/TMJ group. Meal duration was recorded by computer for 24 hours prior to CFA injection, pre-day (0) and 22 days thereafter, post-days (1, 2, 3, etc.). About half of the rats treated with CFA showed additional lengthening in meal durations. Statistical analysis showed that meal duration values (arrow) were significantly longer than the previous day's meal duration. Note brackets comparing meal duration values for specific days where c = P<0.05, d = P<0.001. See the legend of Figure 1 for explanations and abbreviations.

Figure 3

Interleukin 1β (IL-1β) and IL-6 levels in the TMJ tissues of Sprague-Dawley rats. (A) IL-1β or (B) IL-6 levels within the synovial membrane, joint capsule, retrodiscal tissue, articular disc and a small portion of the lateral pterygoid muscle 7, 21 and 42 days after saline or CFA injection. Within a particular day, the SALINE/TMJ group was compared to the CFA/TMJ group and is indicated by e = P<0.01. The letter “f” indicates that the cytokine levels in the CFA/TMJ-treated groups on days 21 and 42 were significantly (P<0.01) reduced compared to the values of the CFA/TMJ-treated group on day 7. See the legend of Figure 1 for explanations and abbreviations.

Figure 4

Calcitonin gene-related peptide (CGRP) levels in the retrodiscal, synovial and disc tissue of the TMJ of Sprague-Dawley rats 7, 21 and 42 days after saline or CFA injection. Within a particular day, the SALINE/TMJ group was compared to the CFA/TMJ group, as indicated by e = P<0.01. See the legend of Figure 1 for explanations and abbreviations.

Figure 5

DBA/1LacJ mice received a bilateral injection of 6 μl saline (n = 12) into the TMJ (SALINE/TMJ) or 30 μg of CFA (n = 11) into the TMJ (CFA/TMJ) or 30 μg of CFA (n = 5) was injected into the knee (CFA/KNEE). A) Dark phase meal duration was calculated for the DBA/1LacJ mice using a 5 minute end-of-the meal definition and a B) 10 minute end-of-the meal definition. C) C57BL/6 mice received bilateral injection of 6 μl saline (n = 7) into the TMJ (SALINE/TMJ) or 30 μg of CFA (n = 5) into the TMJ (CFA/TMJ) and the dark phase meal duration was calculated using an end-of-the-meal definition of 10 minutes. Meal duration was recorded on the day before injection, pre-day (0), and seven days after injection. Values are given as the means ± SEM. A comparison of the SALINE/TMJ group to the CFA/TMJ group is indicated by a = P<0.05 and a comparison of the CFA/TMJ group to the CFA/KNEE group is indicated by b = P<0.05.

Figure 6

IL-1β and IL-6 cytokine levels in the TMJ tissues of DBA/1LacJ and C57Bl/6 mice after CFA injection. IL-1β levels per mg of total protein within the retrodiscal, synovial and disc tissue of the TMJ two days (A) and seven days (C) after CFA injection. IL-6 levels per mg of total protein within the TMJ tissues two days (B) and seven days (D) after CFA injection. Within a particular day when comparing the SALINE/TMJ group to the CFA/TMJ group a significance of P<0.05 is indicated by the letter “a” and a significance of P<0.01 is indicated by the “e”. See the legend of Figure 5 for explanations and abbreviations.