Nociceptive behavioural assessments in mouse models of temporomandibular joint disorders

Abstract

Orofacial pain or tenderness is a primary symptom associated with temporomandibular joint (TMJ) disorders (TMDs). To understand the pathological mechanisms underlying TMDs, several mouse models have been developed, including mechanical stimulus-induced TMD and genetic mouse models. However, a lack of feasible approaches for assessing TMD-related nociceptive behaviours in the orofacial region of mice has hindered the in-depth study of TMD-associated mechanisms. This study aimed to explore modifications of three existing methods to analyse nociceptive behaviours using two TMD mouse models: (1) mechanical allodynia was tested using von Frey filaments in the mouse TMJ region by placing mice in specially designed chambers; (2) bite force was measured using the Economical Load and Force (ELF) system; and (3) spontaneous feeding behaviour tests, including eating duration and frequency, were analysed using the Laboratory Animal Behaviour Observation Registration and Analysis System (LABORAS). We successfully assessed changes in nociceptive behaviours in two TMD mouse models, a unilateral anterior crossbite (UAC)-induced TMD mouse model and a β-catenin conditional activation mouse model. We found that the UAC model and β-catenin conditional activation mouse model were significantly associated with signs of increased mechanical allodynia, lower bite force, and decreased spontaneous feeding behaviour, indicating manifestations of TMD. These behavioural changes were consistent with the cartilage degradation phenotype observed in these mouse models. Our studies have shown reliable methods to analyse nociceptive behaviours in mice and may indicate that these methods are valid to assess signs of TMD in mice.

Fig. 1

The experimental flow and methods used to assess the mechanical allodynia thresholds and bite forces in mice with TMDs. a Experimental flow chart. b A mouse was placed into a cage with a gap at the centre (red arrows). The von Frey filament (green arrow) was passed through the gap and probed against the mouse orofacial skin in the TMJ region (yellow arrow). c A FlexiForce sensor, from Tekscan (left), was passed through the gap and bit by the mouse (right). d, e Representative force–time graphs for β-catenin^Act and Cre^– mice

Fig. 2

OA-like lesions appeared in the mouse TMJ cartilage in 9-week-old mice (3 weeks after the unilateral anterior crossbite (UAC) operation). a Body weights were measured in 7-, 8- and 9-week-old mice (1-, 2-, and 3-weeks post operation) (n = 10). b Decreased cartilage thickness and chondrocytes with reduced Alcian blue/haematoxylin staining were observed after the operation. c The severity of the OA-like phenotype was analysed by assessing the histological grade in the TMJ using the OARSI scoring system (n = 10). d The mouse condylar cartilage area was quantified by tracing the Alcian blue-positive areas using the OsteoMeasure system (n = 10). e Quantitative analysis of changes in the mouse condylar cartilage in the anterior, central, and posterior thirds (n = 10). Statistical analysis was conducted using an unpaired Student’s t test. *P < 0.05, **P < 0.01

Fig. 3

Mechanical allodynia increased in the mouse TMJ region 3 weeks after implanting the UAC prosthesis. a The mechanical allodynia threshold was measured using von Frey filaments in mice 1–3 weeks post operation (n = 10). b The bite force was assessed using FlexiForce sensors in mice 3 weeks post operation when unloading the UAC prosthesis (n = 10). c–e The food intake over 15 h was recorded, and the eating duration and frequency were tested using LABORAS in mice 1–3 weeks post operation (n = 10). Statistical analysis was conducted using two-way ANOVA, followed by the Tukey–Kramer post hoc test. *P < 0.05, **P < 0.01

Fig. 4

β-catenin conditional activation mice (β-catenin^Act) develop osteoarthritis (OA)-like phenotypes in the temporomandibular joint (TMJ). a Body weights were measured in mice at 4, 6, and 8 weeks of age (n = 7). b Representative histology images of TMJ tissue, with Alcian blue/haematoxylin staining. c The severity of the OA-like phenotype was analysed by assessing the histological grade in the TMJ using the OARSI scoring system (n = 7). d The mouse condylar cartilage area was quantified by tracing the Alcian blue-positive areas using the OsteoMeasure system (n = 7). e Quantitative analysis of the changes in the anterior, central, and posterior thirds of the mouse condylar cartilage (n = 7). Statistical analysis was conducted using an unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

β-catenin^Act mice demonstrate increased mechanical allodynia in the TMJ region. a The mechanical allodynia threshold was measured using von Frey filaments at 4, 6, and 8 weeks of age (n = 7). b The bite force was measured using FlexiForce sensors at 4, 6, and 8 weeks of age (n = 7). c–e The food intake over 15 h was recorded, and the eating duration and frequency were assessed using LABORAS at 4, 6, and 8 weeks of age (n = 7). Statistical analysis was conducted using a two-way ANOVA, followed by the Tukey–Kramer post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001