| ObjectiveSkin wound healing is a complex process involving blood clotting, inflammation, granulation tissue formation, re-epithelialization, and finally tissue remodeling. Cannabinoid receptor type 1(CB1R), a main member of endocannabinoid system (ECs), is a G protein-coupled receptor (GPCR). It is predominantly expressed in central nervous system, it also exists in peripheral organs such as liver, gastrointestinal tract. In the present study, the expression and changes of CB1R in incised skin wound healing are detected on the application of two specific polyclonal antibodies to different intracellular domains of CB1R. The investigation is aimed at exploring the possible role of CB1R in skin wound healing and its applicability to the wound age estimation.Materials and MethodsA total of 50 adult, healthy mice of male, each weighting 35-40g, were randomly divided into ten groups, one control group and nine skin incision groups. A 1.5cm-long full-thickness incision deep to the fascia was made with a scalpel in the skin layer on the central dorsum of each mouse under sterile technique after the mouse was anesthetized by inhalating diethyl ether. After injury, each mouse was housed individually and fed with sterilized food and tap water to prevent from infection. The 1.5cm×1.0cm specimens were taken from the wound site after the mice were executed by cervical dislocation at Oh,6h,12h, Id,3d,5d,7d, 10d and 14d postinjury. The control group specimens were taken at the same place by the same way at the 14th day to prepare for the subsequent procedures. The level of CB1R in mouse skin incised wound was detected by immunohistochemical staining and western blotting, and the non-incised mouse skin was used as control. The number and ratio of CB1R-positive cells were counted and analyzed by microscope. The band AverGray was analyzed by Fluochem V2.0. The data were analyzed comparatively by SPSS13.0 for Windows.ResultsCB1R-positive immunoreactivity was found in epidermis, hair follicle, sebiferous gland and dermomuscular layer in normal mouse skin. Expression of CB1R was undetectable in polymorphonuclear cells (PMNs) in the wound specimens at 6h postinjury. In the wound specimens from 12h to 24h postinjury, CB1R-positive immunoreactivity was identified in part of mononuclear cells (MNCs). Afterwards from Id to 3d postinjury, the CB1R-positive cells were mainly found in MNCs and spindle-shaped fibroblastic cells (FBCs) and mainly in FBCs from 5d to 14d postinjury. Morphometrically, the ratios of the number of CB1R-stained MNCs and FBCs to total number of the cells were evaluated and calculated. The ratios of CB1R-positive cells elevated gradually in the wound specimens from Oh to 1d postinjury and kept a high level from 3d to 5d postinjury, with a peak at 5d, and decreased from 7d to 14d postinjury. There were significant differences in ratios of CBlR-positive cells between the neighboring time groups (P<0.05). CB1R-positive band was also noticed in normal mouse skin by western blotting. The changes of CB1R expression were observed throughout the wound healing stages by western blotting as well, with a peak in specimens at 5d postinjury. There were significant differences in avergray of CB1R-positive band between the neighboring time groups (P<0.05), except the difference between Oh group and control group (P>0.05).Conclusions1. CB1R is detected in epidermis, hair follicle, sebaceous gland and dermomuscular layer in normal mouse skin.2. CB1R is found in MNCs and FBCs during the incised skin wound healing in mice, and CB1R may involve in inflammation and skin wound healing. 3. The time-dependent expression of CB1R may be used as a marker for the wound age determination of the incised skin. |
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