The cannabinoid receptor type 2 is time-dependently expressed during skeletal muscle wound healing in rats

Abstract

The expression of the cannabinoid receptor type 2 (CB2R) was investigated by immunohistochemistry, Western blotting, and RT-PCR during wound healing of contused skeletal muscle in rats with attempt of its applicability to skeletal muscle wound age estimation. Furthermore, Macrophage Marker (MAC387) was utilized to identify macrophages recruited into injured skeletal muscle tissue. Co-localization of CB2R with Macrophage Marker was detected by confocal laser scanning microscopy. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, immunoreactivity of CB2R was detected in the sarcolemma and sarcoplasm of normal myofibers. In the contusion groups, a few polymorphonulcear cells, a large number of macrophages, and spindle-shaped fibroblastic cells showed a positive staining for CB2R in wounded zones. By Western blotting analysis, the average of CB2R to GAPDH ratios in 5-7 days post-injury groups was highest, and all the samples had ratios of >2.60. In the other groups, no samples showed ratios of >2.60 and the CB2R to GAPDH ratios ranged from 1.19 to 2.59. The expression tendency was also confirmed by RT-PCR. From the viewpoint of forensic pathology, these observations suggested that the ratio markedly exceeding 2.60 strongly indicated a wound age of 5-7 days. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R be involved in modulating macrophages in response to inflammatory event in rat skeletal muscle wound healing and CB2R be available as a marker for wound age determination.