Evolution of mirror-image pain in temporomandibular joint osteoarthritis mouse model

Abstract

Objective: Mirror-image pain is a kind of pain that occurs on the contralateral side, but its pathogenesis remains unclear. To develop an osteoarthritis mouse model for investigating mirror-image pain through observing nocifensive behaviors, histological changes, and nociceptive activity at days 3, 7, 14, 21, and 28 after the chemical induction of unilateral temporomandibular joint (TMJ) osteoarthritis.

Methodology: We randomly divided 6-week-old mice into sham and complete Freund adjuvant groups. To induce nocifensive behaviors, we applied 0.04 g of von Frey filament, 10 psi of air puff, and cold acetone on both sides of whisker pads at different days. The histology of TMJ on both sides was observed by hematoxylin/eosin staining and microcomputed tomography scanning. Furthermore, the nociceptive activity was evaluated using the phosphorylated cyclic AMP response element binding protein (pCREB) and a microglia marker at different days in the trigeminal subnucleus caudalis.

Results: Nocifensive behaviors against mechanical and temperature stimuli on the contralateral side became stronger than the baseline on day 28, in agreement with the elevation of the pCREB and the microglia marker in the trigeminal subnucleus caudalis. Thus, hypernociception on the contralateral side occurred at day 28.

Conclusions: Clearly, the TMJ model with unilateral osteoarthritis exhibited mirror-image pain. Therefore, this model is useful in investigating the pathogenesis of pain and in developing treatments.

Figure 1. No difference in the bodyweight between the sham and complete Freund adjuvant groups at every time point

Figure 2. Development of mirror-image pain-like behaviors 28 days after the injection of complete Freund adjuvant. The line graph shows the mean pain response score of three stimuli, namely, 0.04 g of Von Frey, cold acetone, and 10 psi of Air-puff tests, for the two mouse groups. *p<0.05: sham vs. complete Freund adjuvant by independent t-test in each time point; n=30 on pre-injection and day 3, n=24 on day 7, n=18 on day 14, n=12 on day 21, and n=6 on day 28.

Figure 3. Injection of complete Freund adjuvant causes anatomical changes of the condylar head of temporomandibular joint. (A) A representative image of microcomputed tomography scans of the temporomandibular joint at days 21 and 28, with scale bars of 5 mm of the upper picture and 1 mm of the inset box. (B) The bar graph shows the mean bone density of the ipsilateral and contralateral sides, * p<0.05: sham vs. complete Freund adjuvant by one-way analysis of variance (ANOVA) and Dunnett’s test (post hoc test) sequentially in each time point, n=6

Figure 4. Representative image of hematoxylin/eosin staining of the condylar head of the temporomandibular joint at days 21 and 28, with a scale bar of 200 µm. The black arrow represents grade 3 at day 21 after complete Freund adjuvant injection, whereas the red arrow represents grade 4 at 28 day after the injection. Both sides for the sham group and contralateral side of the complete Freund adjuvant group obtained grade 0, n=6

Figure 5. Expression of pCREB induced by complete Freund adjuvant on the ipsilateral and contralateral sides of trigeminal subnucleus caudalis. (A) Representative image of pCREB on the ipsilateral and contralateral sides of trigeminal subnucleus caudalis on days 21 and 28, with a scale bar of 100 µm, representing mirror-image pain on the trigeminal subnucleus caudalis. (B) The line graph shows the mean of pCREB expression per section on the ipsilateral and contralateral sides of trigeminal subnucleus caudalis in the sham and complete Freund adjuvant groups on days 3, 7, 14, 21, and 28, *P<0.05: sham vs. complete Freund adjuvant by independent t-test in each time point, n=6

Figure 6. Microglial expression induced by complete Freund adjuvant on the ipsilateral and contralateral sides of trigeminal subnucleus caudalis. (A) Representative image of microglia of the ipsilateral and contralateral trigeminal subnucleus caudalis on day 28, with a scale bar of 100 µm, representing mirror-image pain on the trigeminal subnucleus caudalis. (B) The line graph shows the mean of microglia expression per section on the ipsilateral and contralateral sides of trigeminal subnucleus caudalis in the sham and complete Freund adjuvant groups on days 3, 7, 14, 21, and 28, *P<0.05: sham vs. complete Freund adjuvant by independent t-test in each time point, n=6

Supplementary Figure 1. Threshold of von Frey stimulation (n=20 of naïve mice; mean±standard error of mean)