Histological, histochemical, and protein changes after induced malocclusion by occlusion alteration of Wistar rats

Abstract

Although disorders of the stomatognathic system are common, the mechanisms involved are unknown. Our objective was to study the changes in the masseter muscles after unilateral exodontia. Molar extraction was performed on Wistar rats (left side), and the animals were sacrificed after either 14 or 26 days. The masseter muscle was processed for histological analysis, conventional and in situ zymography, and immunohistochemistry. The morphological analysis showed unique and specific characteristics for the experimental group. By conventional zymography no significant values of 72 kDa MMP-2 (P < 0.05) were found in both of the sides of masseter muscle after 14 and 26 days of unilateral extraction. The in situ zymography showed gelatinolytic activity on all deep masseter muscles, with significant increase on the contralateral side after 14 and 26 days (P < 0.05). The immunohistochemistry demonstrated greater expression of MMP-2 than MMP-9 and MMP-14 in all masseter muscles and there were few differences in the staining of 4 TIMPs. This knowledge about morphology and molecular masticatory muscle remodeling following environmental interventions can be used to develop clinically successful treatments.

Figure 1

Histological analysis with hematoxylin-eosin staining of the contralateral (a, c, and e) and ipsilateral (b, d, and f) masseter muscle. (a, b) Control group. (c, d) Experimental group 14 days after exodontia. (e, f) Experimental group 26 days after exodontia.

Figure 2

Mean ± SD of the average absorbance values obtained for the 72 kDa MMP-2 protein in masseter muscle zymography gels, represented by the following: C, I contralateral and ipsilateral sides of the control group; C14, I14 contralateral and ipsilateral sides after 14 days of exodontia; C26, I26 contralateral and ipsilateral sides after 26 days of exodontia.

Figure 3

In situ zymography of the contralateral (a, c, and e) and ipsilateral (b, d, and f) masseter muscle. (a, b) Control group. (c, d) Experimental group 14 days after exodontia. (e, f) Experimental group 26 days after exodontia.

Figure 4

Quantitative analysis of the gelatinolytic activity found in muscle sections. Data are shown as the mean ± SD. n = 10 per group. *P < 0.05 C versus C14. ^# P < 0.05 C versus C26.

Figure 5

Immunolocalization of MMP-2 in the masseter muscle ((a) contralateral control, (b) ipsilateral control, (c) 14 days contralateral, (d) 14 days ipsilateral, (e) 26 days contralateral, and (f) 26 days ipsilateral), MMP-9 ((ai) contralateral control, (bi) ipsilateral control, (ci) 14 days contralateral, (di) 14 days ipsilateral, (ei) 26 days contralateral, and (fi) 26 days ipsilateral), and MMP-14 ((aii) contralateral control, (bii) ipsilateral control, (cii) 14 days contralateral, (dii) 14 days ipsilateral, (eii) 26 days contralateral, and (fii) 26 days ipsilateral).

Figure 6

Immunolocalization of TIMP-1 in the masseter muscle ((a) contralateral control, (b) ipsilateral control, (c) 14 days contralateral, (d) 14 days ipsilateral, (e) 26 days contralateral, and (f) 26 days ipsilateral) and TIMP-2 ((ai) contralateral control, (bi) ipsilateral control, (ci) 14 days contralateral, (di) 14 days ipsilateral, (ei) 26 days contralateral, and (fi) 26 days ipsilateral).

Figure 7

Immunolocalization of TIMP-3 in the masseter muscle ((a) contralateral control, (b) ipsilateral control, (c) 14 days contralateral, (d) 14 days ipsilateral, (e) 26 days contralateral, and (f) 26 days ipsilateral). Immunolocalization of TIMP-4 in the masseter muscle ((ai) contralateral control, (bi) ipsilateral control, (ci) 14 days contralateral, (di) 14 days ipsilateral, (ei) 26 days contralateral, and (fi) 26 days ipsilateral).