Immobilization induced prolonged inflammatory conditions in rat knee capsule

Purpose: Joint immobilization often induces joint stiffness, however, it is performed in daily examination in orthopaedics. Recent studies suggested that the joint capsule was one of the main sources of joint stiffness. In our previous reports, joint immobilization in rat knee joints induced increased stiffness of the capsule, and up-regulated gene and protein expressions in the capsule of fibrotic factors, such as TGF-β1 and CTGF. Though the precise etiology of joint stiffness has been still unknown, prolonged inflammation is one of the possible causes. In this study, we examined the histological changes of the capsule and expression of inflammatory cytokines by quantitative PCR and immunohistochemistry. Methods: Experimental Design: Unilateral knee joints were immobilized at 150° of flexion with a plastic plate and metal screws for1, 3 days, 1, 2, 4, 8, 16 weeks. Only screws were inserted for sham-operated rats. The immobilized rats and sham-operated rats made up the immobilized group and the control group, respectively. One hundred and twelve rats were prepared for histological analysis (n = 8/each period). Thirty rats were prepared for quantitative PCR (1 day, 1 week, and 4 weeks; n = 5/each period). Histology & Immunohistochemistry: The rats were fixed with 4% PFA in 0.1M PBS and the resected knee joints were decalcified in 10% EDTA in 0.01M PBS and embedded in paraffin. Five-μm sections were obtained at the medial mid-condylar region of the knee in the sagittal plane. The sections were stained with Elastica-Masson and the number of cells per unit in the capsule was counted. The sections were stained with rabbit anti-rat IL-6 antibody (Abcam, ab6672) and rabbit anti-rat IL-1β (Ab Frontier, LF-PA50204), and the expression patterns in the capsule were examined. Quantitative PCR: The anterior and posterior capsule were harvested and immediately placed in 1 ml QIAzol (Qiagen). The tissue was homogenized, the total RNA was purified (RNeasy Lipid Tissue Kit, Qiagen), and the cDNA was synthesized (cDNA Synthesis Kit, Invitrogen). The relative expression levels of IL-6, IL-1α, IL-1β, TNF-α, and TNF-β as a function of EF1α1 was calculated. Results: Histology: The number of cells in the control groups was almost unchanged throughout the experimental periods. The number of cells at 1 day was not changed, however, the number at 3 days was significantly higher in the immobilized group. The number of cells in the immobilized groups peaked at 3 days and then gradually decreased after 1 week. Immunocytochemistry: The staining intensity of IL-6 in the capsule was weak at 1 day, however, the intensity was stronger at 3 days in the two groups. The staining intensity in the control groups peaked at 3 days, and decreased after 1 week. The strong staining intensity in the immobilized groups was kept until 4 weeks, and then became weaker at 8 weeks. The staining intensity of IL-1β was similar fashion to IL-6. Quantitative PCR: Increased expression of IL-6 was observed at 1 day in the two groups. The expression levels in the control groups were normalized after 1 week, whereas, the expression levels in the immobilized groups were kept higher level at 1 week. Conclusions: The prolonged inflammatory conditions were kept in the joint capsule after immobilization. Increased number of cells in the immobilized group may support the results. It was indicated that prolonged inflammation and subsequent fibrosis of the joint capsule was a main pathology of the joint contracture after immobilization.