Effects Of Chronic Sleep Disturbance On The Cartilage Angiogenesis In Temporomandibular Joint Osteoarthritis Of Rats

Objective:Temporomandibular joint osteoarthritis (TMJ-OA) is a kind of sever Temporomandibular disorders(TMD). While the pathogenesis of TMJ-OA is unclear. The aim of this study was to investigate whether chronic sleep disturbance can lead to temporomandibular joint osteoarthritis (TMJ-OA), and examine the vascular invasion in the TMJ. We also wanted to find the regulating mechanism of vascular endothelial growth factor (VEGF), Delta-like 4 (D114) and phosphated-ERK1/2 (p-ERKl/2) promotes TMJ-OA cartilage angiogenesis.Methods:The 180 male rats were randomly divided into three groups (n= 60 per group): the control (CON) group, chronic sleep disturbance (CSD) group and the recover (R) group. Each group was divided into three subgroups. Rats were subjected to chronic sleep disturbance for 4 weeks,6 weeks, or 8 weeks using the modified multiple platform method (MMPM). the serum levels of corticosterone (CORT) and adrenocorticotropic hormone (ACTH) were tested to assess the status of rats. Histomorphology of the TMJ were observed. Immunofluorescence of CD 105 and the microvessel density (MVD) were used to test the angiogenesis in the TMJ of Rats. The VEGF, D114 and p-ERK1/2 expression levels were detected by immunohistochemistry, Western blot analysis or real-time quantitative polymerase chain reaction (PCR). Rat mandibular condylar cartilage (MCC) cells were isolated from normal condyle cartilage of Wistar Rats, treated with VEGF or U0126. Then we examined the relationship of p-ERK1/2 and D114 expression.Results:We successfully established a CSD rat model using the modified multiple platform method (MMPM), a well-established method for introducing sleep disturbance in rats. CSD increases Serum Corticosterone (CORT) and Adrenocorticotropic Hormone (ACTH) Concentrations in the Rat. Comparaed with CON group, The CORT and ACTH Concentrations significantly increased (p<0.05).The results of Haematoxylin and eosin (HE) staining showed that the TMJ appeared normal in the CON group. While OA pathological alterations were found in the Rat TMJ of CSD group and R group. In addition, both the number and score of cartilage damage increased in the CSD and R group to controls (p<0.05).The results of immunofluorescence and HE staining showed that CSD can promote Angiogenesis in the TMJ. We found an increased number of blood vessels in the Osteochondral boundary of TMJ in the CSD and R groups. The MVD of CSD and R groups markedly increased to controls(p< 0.05).Immunohistochemistry showed that while the CON group had no or very low numbers of VEGF-positive-chondrocytes, D114-positive-chondrocytes and p-ERK1/2-positive-chondrocytes. There was an increase in the number of VEGF-positive-chondrocytes, D114-positive-chondrocytes and p-ERK1/2-positive-chondrocytes in the CSD and R groups. We also detected the expression of VEGF, p-ERK1/2 and D114 in TMJs by western blot and RT-qPCR. The expression of VEGF, p-ERK1/2 and D114 markedly increased over time in the CSD group compared with controls (p<0.05).We investigated whether D114 expression was dependent on the activation of p-ERK1/2 in MCC cells. We found that treatment with the MEK/ERK inhibitor U0126 significantly suppressed the expression of D114 induced by VEGF as determined by western blot.Conclusions:We revealed the pathological changes in the TMJ of rats exposed to CSD. Our findings provide important new evidence indicating that CSD in rats causes significant OA pathological changes in the TMJ. And angiogenesis in the osteochondral junction is an important factor in the pathogenesis of TMJ-OA. We found increased expression of VEGF, Dll4 and p-ERK1/2 in the MCC cells. Moreover, we showed that VEGF-induces p-ERK1/2 signaling, which in turn, increases D114 expression and promotes angiogenesis in the TMJ.