Dynamic localization of CB2R and quantitative analysis of CB2R mRNA during skin wound healing in mice were performed. Co-localization of CB2R with F4/80 or α-SMA was detected by double-color immunofluorescence microscopy. A total of 110 male mice were divided into control, injury, and postmortem groups. Sixty-five mice were sacrificed, followed by sampling at 0.5 h-21 days post-injury. Five mice without incision were used as control. The other 40 mice that received incised wound were sacrificed at 5 days after injury. The samples were collected at 0 h-3 days postmortem. In the uninjured controls, CB2R immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands, dermomuscular layer, and vascular smooth muscle. In the incision groups, polymorphonulcear cells, macrophages, and myofibroblasts showed positive staining for CB2R. Morphometrically, the average ratios of CB2R-positive cells were more than 50 % at 5 days post-wounding, whereas it was <50 % at the other posttraumatic intervals. The average ratios of CB2R-positive macrophages maximized at 3 days post-wounding, and the average ratios of CB2R-positive myofibroblasts peaked at 5 days post-wounding. The relative quantity of CB2R mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >4.10, which was also confirmed by Western blotting. There was no significant change for CB2R protein within 6 h postmortem and for mRNA within 3 h postmortem as compared with the control group. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R is involved in modulating macrophages and myofibroblasts in response to inflammatory event and repair process in mouse skin wound healing, and CB2R is available as a marker for wound age determination.

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.

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Recent studies have shown that nicotinic acetylcholine receptor alpha7 subunit (nAChRα7) plays an important role in regulation of inflammation, angiogenesis and keratinocyte biology, but little is known about its expression after the skin is wounded. A preliminary study on time-dependent expression and distribution of nAChRα7 was performed by immunohistochemistry, Western blotting and RT-PCR during skin wound healing in mice. After a 1-cm-long incision was made in the skin of the central dorsum, mice were killed at intervals ranging from 6 h to 14 days post-injury. In uninjured skin controls, nAChRα7 positive staining was observed in epidermis, hair follicles, sebaceous glands, vessel endothelium and resident dermal fibroblastic cells. In wounded specimens, a small number of polymorphonuclear cells, a large number of mononuclear cells (MNCs) and fibroblastic cells (FBCs) showed positive reaction for nAChRα7 in the wound zones. Simultaneously, nAChRα7 immunoreactivity was evident in endothelial-like cells of regenerated vessels and neoepidermis. By morphometric analysis, an up-regulation of nAChRα7 expression was verified at the inflammatory phase after skin injury and reached a peak at the proliferative phase of wound healing. The expression tendency was further confirmed by Western blotting and RT-PCR assay. By immunofluorescent staining for co-localization, the nAChRα7-positive MNCs and FBCs in skin wounds were identified as macrophages, fibrocytes and myofibroblasts. A number of nAChRα7-positive myofibroblasts were also CD45 positive, indicating that they originated from differentiation of fibrocytes. The results demonstrate that nAChRα7 is time-dependently expressed in distinct cell types, which may be closely involved in inflammatory response and repair process during skin wound healing.