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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes how measuring bite force and head withdrawal threshold can capture pain behavior in Complete Freund's Adjuvant (CFA)-induced inflammatory temporomandibular joint (TMJ) pain.

Abstract

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms driving diseases-related pain are poorly understood, and current treatments fail to provide effective and long-term therapeutic effects. Additionally, pain assessment in research, particularly orofacial pain, poses several challenges that complicate studies in both clinical and basic science settings. Therefore, we have established an inflammatory TMJ pain mouse model via intra-articular injection of CFA (Complete Freund's Adjuvant) and evaluated pain behaviors by bite force measurement and the von Frey filament test. Mice with CFA injection exhibited orofacial pain behaviors compared to PBS injection, including reduced bite force and head withdrawal threshold in the von Frey filament test. These methods are relatively easy to execute to have reproducible results and can be potentially extended to pain studies for other disease models related to TMJ disorders. Together, bite force, and the von Frey filament tests are reliable in measuring orofacial pain, as demonstrated in CFA injection-induced painful TMJOA mouse models.

Introduction

Temporomandibular disorders (TMD) refer to pain and dysfunction of musculoskeletal and neuromuscular structures affecting the temporomandibular joints (TMJ), masticatory muscles, and associated structures. The TMJ is the most frequently used synovial joint in daily life. About 80% of TMD patients show signs and symptoms of TMJ diseases, which can significantly impact their quality of life1,2,3. In addition, TMJ osteoarthritis (TMJOA) is an important subtype of TMD and is defined as a progressive degenerative joint disease. Patients with TMJOA often present with joint pain. Painful TMJ is one of the primary reasons for patients to seek treatment4,5. However, there is an unmet medical need for more effective treatments to alleviate painful TMJOA. Understanding painful TMJOA is crucial for developing disease-modifying OA drugs (DMOADs).

Since obtaining clinical samples from patients with TMJOA pain is difficult, animal models have become essential tools for studying TMJOA pathologies6,7. Integrating the assessment of pain behavior can provide valuable insights into its underlying mechanisms and potential new therapeutic interventions. Pain measurement in animal models is challenging due to the subjective nature of pain perception and expression in non-human subjects6,8. Relatively few studies have used pain behavioral assays in TMJ compared to non-craniofacial joints. Therefore, it is important to define methods for assessing pain behavior to reliably capture the pain experience in animal models of TMJOA.

In this article, we have illustrated two distinct methods designed to evaluate pain in mouse models of TMJOA: bite force measurement and the von Frey filament test. Jaw and muscle coordination is essential for masticatory function, and pain associated with TMJ and muscle function can reduce bite force in TMD patients3,9. Thus, pain-related behavior secondary to masticatory function can be assessed using bite force measurement, which effectively reflects TMJ function related to joint pain. Additionally, the von Frey filament test measures the head withdrawal threshold to assess mechanical sensitivity. In this method, von Frey filaments with increasing force are applied to the skin of mice until a withdrawal response is observed. The force that triggers the head withdrawal response is recorded as the head withdrawal threshold. This technique is widely used for assessing mechanical allodynia, indicating peripheral nociceptor sensitization associated with orofacial pain8,10,11.

Although previous studies have used these methods to measure pain behavior, none have provided detailed protocols for direct implementation or combined these methods to assess TMJ pain induced by intra-articular injection of CFA12,13,14,15. Therefore, these methods are developed and validated to demonstrate their sensitivity and reproducibility in measuring the severity and progression of pain. These two complementary approaches provide cross-validation for pain behavior assessment. Importantly, the pain behavioral outcomes from these techniques can correlate with the histological and pathological features in painful TMJOA mice14,16. Additionally, measuring bite force provides valuable data for evaluating jaw function, while the von Frey test measures mechanical nociception8. Therefore, these protocols can be applied to assess pain behavior in various orofacial pain conditions, including odontogenic pain, TMJ pain, muscle pain, and neuropathic pain.

Protocol

All animal procedures were approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC). Eight-week-old C57BL/6 female mice with body weight between 19-20 g were used in this study. The mice were housed in standard cages under environmental conditions with regulated humidity and 12-h light/dark cycles. The reagents and equipment used are listed in the Table of Materials.

1. CFA intra-articular injection to induce TMJOA

  1. Anesthetize the mice with isoflurane using 3% isoflurane for induction and 1%-2% for maintenance (following institutionally approved protocols). Use a nosecone for the entire procedure to maintain anesthesia. Perform a toe pinch to verify sufficient anesthetic depth.
  2. Closely monitor the mice's breathing to ensure proper respiratory status while minimizing their exposure to anesthesia. Apply eye gel to protect the eyes during the procedure.
    NOTE: The mice could not receive analgesia for post-surgical pain in order to avoid pre-emptive analgesic effects on the development of orofacial pain and the measurement of pain behavior.
  3. Prepare a sterile insulin syringe or a 31 G needle for injecting Complete Freund's Adjuvant (CFA) 5 mg/mL into the TMJ by covering the needle with a sheath made of sterile polyethylene tube, leaving only 2 mm of the needle tip protruding, to prevent it from being inserted too deeply.
  4. Remove the hair and disinfect the preauricular area with povidone-iodine and 70% alcohol after anesthesia.
  5. Identify the zygomatic arch by palpating it to locate the TMJ injection site.
    NOTE: At the posterior end of the zygomatic arch, there is a depressed area approximately 2 mm in front of the ear canal. The TMJ injection site is located around this area and should not extend beyond the imaginary line drawn from the level of the eye to the ear canal (Figure 1A,B).
  6. Insert the needle into the injection site, which is under the posterior end of the zygomatic arch. The tip of the needle should contact the bone at a depth of 2 mm (Figure 1A,E).
  7. Slowly inject 10 µL of CFA into the TMJ capsule and wait for at least 5 s before gradually withdrawing the needle. Inject the same amount of PBS into TMJ in the control mice following the same procedure.
  8. Repeat steps 1.4-1.8 and inject CFA into the opposite TMJ. Observe the mice on the heat pad until they are fully recovered.
    NOTE: CFA is a water-in-oil emulsion that is relatively viscous, therefore, CFA injection and needle withdrawal should be slow to avoid CFA leaking during injection. To ensure the precision of injection, practice injection by administering 5 µL fast green dye followed by dissecting masseter muscle to examine the injection accuracy (Figure 1D,E). After injection, fast green dye diffuses rapidly, making it necessary to reduce the amount used. This reduction not only improves injection precision but also prevents unwanted diffusion into neighboring areas. The amount and concentration of CFA administered for inducing TMJOA in mice were determined based on the protocol established in previous studies16,17.

2. Measurement of bite force

  1. Acclimate the mice to the testing environment by placing the tube designed to restrain them in the mouse cage for at least 1 h in the behavioral room before testing.
  2. Record the baseline measurement of bite force before starting to induce inflammation. Train the mice to bite within the testing environment for 3 days before CFA or PBS injection. Record the average values as baseline training values.
  3. Turn on the compatible software to measure bite force using a bite force transducer that is connected to the program on the computer (Figure 2A,B).
    1. Open the NBIT RSD-V2.6.3 program to show the oscillograph (Figure 2C).
    2. Select proper communication options as follows: Attended Mode: UDP (Figure 2E). Select the appropriate Local IP, and search Device IP and Port by clicking on Search. Target IP and Target Port will show up, then click on Open.
    3. Set the Y axis by clicking on 10 in Range Y. Then click on Set (Figure 2F) . Click on Acquisition.
    4. Select only Channel 1 (Ch1) for the blue graph and turn off other channels that are not connected to the device (Figure 2D).
    5. Click on Play to start recording bite force (Figure 2F).
      NOTE: Before clicking "Play "to start recording, the mouse should be already inside the syringe and ready to record bite force in order to get an accurate recording time.
  4. Put the mouse in a 50 mL plastic tube, which has been modified to loosely restrain the mouse. A wide opening on one side is specifically designed for the mouse's head to move comfortably.
  5. Move the bite plate towards the mouth.
    NOTE: The mice will automatically start biting once the bite plate touches their mouth. However, it could take about 30 s for the mice to start biting in the first training. After training, the mice should start to bite within 10 s. If the mice do not start biting within 10 s after the third training day, they will be excluded from the experiment. Thus, training the mice to become familiar with the testing environment is important.
  6. Record the voluntary biting for 2 min/session. Then, click on Pause to stop recording and click on Save (Figure 2E). The results from bite force recording will be automatically exported to an Excel file (Supplementary File 1).
    NOTE: In the Excel file (Supplementary File 1), the data in the left column represents the time at recording. The second column from the left is the amplitude of bite force at each time point.
  7. Rank the data in the second column in the Excel file from maximum to minimum. Select the top five of bite force amplitude for further analysis.
    NOTE: The top five amplitudes should be at different time points, for example, every 20 s or 1000 of the recording time, to prevent selecting the data from the same event of biting.
  8. Record the Value of Ch1 (Figure 2D) from the program and subtract or add to the top five values of bite force amplitude to get the relative values of bite force from this assay. Then, take an average of these five values to get the average relative bite force for one mouse.
  9. Repeat steps 2.3.5-2.8 to record bite force for the next mice.
    NOTE: To assess the bite force in the progression of TMJ pain, the values of relative bite force are recorded during the baseline training and measured at different time points (such as Day 1st, 3rd, 5th, 7th, and Day 12th) after CFA injection.

3. Measurement of head withdrawal threshold

  1. Put a wire mesh cage in the mouse cage and habituate the mice in a behavioral room for at least 1 h before the testing procedures.
  2. Train the mice within the testing environment and record the baseline head withdrawal threshold for 3 days before CFA or PBS injection.
  3. Turn on the electronic von Frey test machine to measure the head withdrawal threshold (Figure 3A,B).
    1. Press MAX and then set the unit to 500 g, which is an arbitrary unit set by the machine.
    2. Set zero to start recording. The value of force on the screen should be at zero (Figure 3C,D).
  4. Place mice in the wire mesh cage (Figure 3B). Apply the von Frey filament perpendicularly to the TMJ area.
    NOTE: The test area is at the level of the connection between the eyes and the ear canal, approximately 2 mm in front of the ear canal. The head withdrawal threshold is defined as the minimum force from the von Frey filament, which can elicit a withdrawal reflex.
  5. Record the value of force applied from the filament or the stimulus intensity (g) that triggers the reactions as the first test (Figure 3E).
  6. Set the value of force on the screen to Zero and wait for 10 s while the mouse is still inside the cage.
  7. Start applying the filament again and record the value.
  8. Repeat the measurement (steps 3.5-3.7) and record at least five tests for each mouse at an interval of 10 s.
  9. Take an average of these five values. The average value is considered as the head withdrawal threshold from one mouse.
    NOTE: Von Frey filament measurement will be performed 5 times for one mouse. If there is a robust variation, measurement can be repeated up to 8 times without notable injury. Either bite force or the von Frey test can start first. There is no significant difference in terms of which test starts first based on our experience. However, it should be consistent during the whole experiment. The interval time between bite force and von Frey is at least 30 min. After the first assay, the mice were put back in the cage. Let them rest for 30 min and start the second assay.

Results

Inflammatory-induced TMJOA pain is induced by injecting CFA into the TMJ. As the left and right TMJ must coordinate during jaw movement, CFA is injected into both the left and right joints to assess TMJ pain. The sham group will receive injections of PBS as controls. Mean ± SEM and Student's t-test are used to statistically analyze the differences in pain behavior between the CFA and sham groups.

Measurements of bite force and head withdrawal threshold are used to examine whether mice experience TMJ pain. CFA-treated mice exhibited significant reductions in both their bite force levels and head withdrawal thresholds on day 1 following CFA injection compared to their baseline levels, with gradual increases observed at later time points. In contrast, the control group injected with PBS showed no significant changes in bite force or responses to von Frey filament tests compared to their baseline training (Figure 4A-D). These results suggest that mice receiving CFA injections experienced TMJ pain with mechanical allodynia and impaired masticatory function.

figure-results-1275
Figure 1: TMJ intra-articular injection in mouse model. (A) The mouse was anesthetized with isofluorane using a nosecone. The yellow line indicated the imaginary line from eye level to ear canal. (B) Anatomical landmark for injection. The black dash line indicates the zygomatic arch, which can be palpated from the skin. The depressed area for the injection site was indicated with a red mark. (C) The condylar head of TMJ can be seen when removing the masseter muscle. (D,E) Fast green dye was injected to indicate the condylar head of TMJ. Scale bar: 1 mm. Please click here to view a larger version of this figure.

figure-results-2264
Figure 2: Recording of bite force measurement. (A,B) Bite force machine and the computer for recording bite force. (C) The amplitude of the bite force recording during bite force measurement. (D-F) Parameters in the program for recording bite force. (D'-F') Enlargements of each parameter section. Please click here to view a larger version of this figure.

figure-results-3084
Figure 3: Electronic Von Frey analgesiometer. (A,B) Von Frey filament instruments. (C) The screen that shows up when the machine is turned on. (D) Set the machine to zero to start recording. (E) The amount of force applied by von Frey to trigger head withdrawal response. Please click here to view a larger version of this figure.

figure-results-3785
Figure 4: Pain behavior assessment in CFA-induced inflammatory TMJOA compared to PBS control group. (A,B) Bite force measurement and the percentage change of bite force values compared to baseline. (C,D) Measurement of head withdrawal threshold by von Frey filament test and the percentage change of head withdrawal threshold compared to baseline. Values represent mean ± SEM, Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 6 mice for PBS group, n = 8 mice for CFA group. Please click here to view a larger version of this figure.

Supplementary File 1: Bite force measurement and analysis. Column A indicates the time point of each bite event, while Column B records the amplitude or value of the bite force. This table presents data organized by the maximum absolute value of the bite force. Please click here to download this File.

Discussion

An inflammatory-induced TMJOA in mice serves as an animal model to investigate pain mechanisms in TMJOA since the intra-articular CFA injection can induce pain and inflammation, resulting in synovitis, bone loss, and cartilage remodeling. These conditions resemble the pathology observed in humans to a certain degree and contribute to the main characteristics of TMJOA, such as joint pain and inflammation in patients13,18. Painful symptoms associated with TMJOA are the major reason for patients to seek medical help. Being able to detect the presence and severity of pain in animal models provides opportunities to investigate pain pathogenesis and potential new treatments. This report described the method for assessing TMJ pain behavior through measurements of bite force and head withdrawal threshold. TMJ is one of the key structures involved in all jaw movement, including mouth opening-closing, biting, and chewing. Additionally, the dominant clinical manifestation of TMD is pain associated with chewing or mastication. TMD patients with TMJ pain typically have reduced bite force compared to healthy individuals9,19. Consistent with clinical studies, intra-articular injection of CFA in mouse models can lead to a significant decrease in bite force that could be considered equivalent to symptoms related to TMJ pain and dysfunction in humans11.

The clinical diagnosis of TMJ pain involves palpating the TMJ and recording the severity of pain based on the patient's report. This procedure is similar to the von Frey filament test, which measures the lowest threshold force leading to head withdrawal behavior. Head withdrawal indicates pain response and hyperalgesia state of the orofacial region20. A reduced head withdrawal threshold may indicate joint tenderness and a lower pressure pain threshold in patients with TMJ pain18,19,21. Consequently, the von Frey filament test is useful for assessing the pressure pain threshold for TMD. In the CFA-induced TMJOA mouse model, measuring bite force and head withdrawal threshold has been validated as a reliable method for evaluating pain behavior22.

Most behavioral outcomes are somewhat subjective. Like many other behavioral assays, variation is always a concern when measuring orofacial pain. To achieve the goal of reliable, reproducible, and sensitive measurements of TMJ pain, it is important to note the limitations and potential solutions in our experimental design, execution, and result interpretation. One caveat is that reduced bite force could be caused by TMJ dysfunction that is not necessary from joint pain. For example, muscles play a crucial role in TMJ movement and biting. Reduced bite force could be due to muscle dysfunction rather than joint pain. However, radiographic and histological findings can be used to differentiate CFA-induced TMJ pain from muscle pain. TMJ pain from CFA injection will subsequently lead to cartilage and bone loss, whereas CFA-induced muscle pain is less likely to cause bone loss16.

It should be noted that this study used electronic von Frey to measure the head withdrawal threshold on a continual scale, which can minimize the number of force applications and thus prevent injury to the facial skin in the test area23. It has been reported that there are notable differences in the withdrawal threshold values obtained from manual and electronic von Frey. Typically, the withdrawal threshold measured with electronic von Frey tends to be higher than that measured with manual von Frey8,10. The plastic tip of our electronic von Frey is not sharp to avoid facial skin injury, which could lead to different values from manual von Frey filaments. In addition, the "g" unit in the electronic von Frey test is not an actual force but an arbitrary unit set by the machine. Additionally, to minimize the skin pain, the hair was removed from the injection site before starting baseline training. This approach should reduce the confounding factors of skin pain and sensitivity and prevent potential infection contamination. Meanwhile, PBS was also injected in the sham group under the same procedure to maximally exclude the confounding effects from skin pain and to ensure the measurement is from joint pain. Although intra-articular injection of CFA is specific to the TMJ area, CFA-induced tissue inflammation is likely not limited to the joint itself, which potentially contributes to some pain response, including reduced bite force and decreased head withdrawal threshold. Overall, we acknowledge that we cannot completely exclude the secondary effect of TMJ inflammation-derived skin or muscle pain, which might contribute to the overall outcome.

The values of bite force and head withdrawal threshold obtained from each instrument could be different since they have different units, parameters, and protocols that are specifically set up for individual assays. Although female mice were used in this study, males can also be used to study painful TMJOA. However, there is sexual dimorphism in the basal nociceptive thresholds of rodents. Males and females exhibited various pain experiences, which can be characterized by distinct physiological and behavioral responses to painful stimuli24,25,26. Additionally, male mice tend to have a higher head withdrawal threshold and bite force than females. Age and body weight also affect bite force values11,16,27. A previous study reported that there was a positive correlation between bite force and body weight during the initial two weeks of observation11. Bite force values for female C57BL/6 mice at 8 weeks old at baseline in this study ranged from 4.5-5.5 N (Figure 4A). After CFA injection, the reduction in bite force to 2-3 N in the early phase is consistent with the previous studies involving the induction of TMJOA using the MIA inflammatory substance6. However, using the percentage change in bite force can serve as a means to validate the consistency of findings with other previous studies since each laboratory might have different instruments and protocols, resulting in different values27,28.

To reduce variation in pain behavior measurement, the critical step is to validate and ensure consistency in training the mice at baseline. Variation can arise from differences in sex, body weight, and age, all of which can affect bite force and head withdrawal threshold measurements. For example, 8-week-old female mice typically weigh around 19-20 g; relative bite force values usually fall within the range of 4.5-5.5 N based on data from baseline training. Therefore, mice with average bite force values outside baseline training ranges should be excluded from the experiment. Similarly, the head withdrawal threshold during baseline training ranges between 22-35 g. Mice with a head withdrawal threshold outside this range should be excluded from the experiment. Overall, it is important to establish the benchmark of bite force and head withdrawal threshold during animal training before the formal behavioral tests.

Disclosures

The authors declare there are no competing financial interests that might be perceived as affecting the objectivity of these studies.

Acknowledgements

We thank the Chen laboratory colleagues for stimulating discussions. We appreciate the support from Thach-Vu Ho for helping with mouse images. This study was supported by grant R01DE033511 (J.C.) from the National Institute of Dental and Craniofacial Research (NIDCR).

Materials

NameCompanyCatalog NumberComments
Bite force transducer Nanjing Shen-yuan-sheng Intelligent Technology CoNBIT-YFM-1-1000–100 N, a bite force transducer connected to the NB IT RSD-V2.6.3 program (NST2000 data collector) from Nanjing Shen-yuan-sheng Intelligent Technology Co., PR. China 
Complete Freund's Adjuvant (CFA)Chondrex Inc7023CFA 5mg/mL
IsofluoraneFluorisoMWI501017
NBIT RSD-V2.6.3 programNanjing Shen-yuan-sheng Intelligent Technology Co
Polyethylene tubeScientific Commodities IncNC0923369PE Tubing 0.38 x 1.09 mm 100 Ft
Von Frey analgesiometerBiosebBIO-EVF4Electric Von Frey

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