Joint hemorrhage accelerated joint contracture in immobilized knee in rats

Purpose: Joint immobilization is commonly used as a treatment for joint injuries and diseases. However, it also causes unfavorable outcomes such as joint contracture. In our previous reports, joint immobilization caused adhesion and shortening of the joint capsule and restricted range of motion. Joint hemorrhage occurs by intra-articular fractures, ligament ruptures and haemophilia. Some researchers have reported blood-induced joint damage using animal models. Although influence of blood on a synovial membrane (SM) and cartilage matrix was reported, the precise mechanism is still controversial. The purpose of this study was to elucidate capsular changes after single blood injection in immobilized knees in rats. Methods: Animals: The unilateral knee joints of Sprague-Dawley rats aged 12-week old were immobilized at 150° of flexion with a plastic plate and metal screws for various periods (1, 3 days, 1, 2, 4, and 8 weeks). Sham operated rats had holes drilled in the femur and tibia with screws, but the plate was not inserted. After the operation, the rats were divided into three groups: Immobilized-blood injection (Im-B) group, Immobilized-normal saline injection (Im-NS) group, and Sham-blood injection (Sm-B) group. Fifty μl of autologous blood were administered intra-articularly for the Im-B and Sm-B groups just after the surgery. The same amount of normal saline was administered for the Im-NS group. Histology & Immunohistochemistry (IHC): Paraffin embedded 5-μm thick sagittal sections in the medial midcondylar region of the knee were made. The sections were stained with Elastica-Masson to observe morphological changes of the SM and the capsule, and with Perls’ Prussian blue to visualize iron deposition in the SM and capsule. The expression patterns of CD68, TGFβ1, and collagen types I and III in the capsule were evaluated by IHC. Scanning acoustic microscope (SAM): In general, sound speed is in proportion to the square route of Young's elastic modulus. SAM can measure sound speed of tissues on slide glass in situ. We set the region of interests and their average sound speed was calculated with a gray scale SAM images with image analysis software. Results: Histology & IHC: Absorption of the injected blood was delayed and made severe adhesions in the Im-B group (Fig. 1A-C). Shortening of SM was observed in the capsules due to adhesion of the opposing capsule, the articular cartilage, and the meniscus. The length of posterior SM in the Im-B group was significantly shorter than that of the other groups from 1 to 4 weeks (Fig.1D and E). The iron deposition in the capsule was observed in the Im-B and Sm-B groups (Fig. 2A-C). Strong immunoreactivity of CD68 and TGF-β1 were observed in the adhesion areas in the Im-B group (Fig.2D- I). However, the staining intensity of collagen types I and III did not change in the Im-B group compared to the other groups. SAM: The low sound speed areas decreased and high sound speed areas increased in the posterior capsule in the immobilized group (Im-B and Im-NS) (Fig.3A- C). The sound speed of posterior capsule in the Im-B group was significantly higher than that in the Sm-B group (Fig. 3D and E). Conclusions:These data indicated that joint immobilization and blood injections caused irreversible capsular changes. Joint hemorrhage is a risk factor for joint contracture, and drainage of the blood or short immobilization periods might be a good strategy to avoid joint contracture. View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)