| ObjectiveSkin incised wound healing is a complex process, including clotting of blood components, inflammation, granulation tissue formation, re-epithelialization, connective tissue retraction and fibroplasia. The parasympathetic neurotransmitter acetylcholine is synthesised and secreted by non-neuronal cells (keratinocyte, endothelial cell, melanocyte et al) in an autocrine, paracrine and endocrine fashion which influenced a plethora of cutaneous basic functions, such as cell proliferation, differentiation, adhesion and migration, microcirculation, angiogenesis, as well as a great deal of immune responses. Experiments demonstrate the non-neuronal cholinergic system and its components expresse in neutrophil, mononuclear macrophage, fibroblast, keratinocyte and endothelial cell which participate in skin incised wound healing. Experiments show M3 subtypes of muscarinic receptor take part ininflammatory reactions and tissue and organ fibrous degeneration, In the present study, the level of M3R in incised skin wound was detected by immunohistochemical technique and Western blot. The investigation is aimed at exploring the possible role of M3R in skin wound healing and its applicability to the wound age estimation.Materials and MethodsA total of 50 adult, healthy mice of either sex, each weighting 35-40g, were divided into ten groups randomly, 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 diethylether. After injury, each mouse was housed individually and fed with sterilized food and distilled water to prevent from infection. The 1.5cm×1.0cm specimens were taken from the wounded site after the mice were executed by cervical dislocation at Oh,6h,12h, 1d,3d,5d,7d, 10d and 14d post-injury to prepare for the subsequent procedures. The level of M3R in mouse skin incised wound was detected by immunohistochemical staining and Western blot, and the non-incised mouse skin was used as control. The number and ratio of M3R-positive cells were counted and analyzed by microscope. The band average optical value was analyzed by software of Scion image. The data were analyzed comparatively by the software of SPSS 13.0 for Windows.ResultsImmunoreactivity of M3R was detected in epidermis, hair follicle, sebiferous gland and dermomuscular layer in normal mouse skin. Expression of M3R was detectable in polymorphonuclear cells (PMNs) in the wound specimens aged 6h. In the wound specimens aged from 12h to 24h after injury, M3R-positive immunoreactivity was identified in a large number of infiltrating PMNs and part of mononuclear cells (MNCs). Afterwards from Id to 3d post-wounding, the M3R-positive cells were mainly found of MNCs and spindle-shaped fibroblastic cells (FBCs) and mainly in FBCs from 5d to 14d post-injury. Morphometrically, the ratios of the number of M3R-stained PMNs, MNCs and FBCs to total number of the cells in the wounds were evaluated and calculated. The ratio of the M3R-positive cells was low in the wounded specimens aged from Oh to 6h, and elevated in the wound specimens aged 12h and kept a highly level from Id to 5d post-injury, with a peak at 12h. Thereafter, the ratios decreased from 7d to 14d and minimized in the specimens aged 14d, with the second peak at the 7d. There were significant differences in ratios of M3R-positive cells between the neighboring time groups (P<0.05) except that between Oh post-injury and normal group.Conclusions 1. M3R is dectected in epidermis, hair follicle, sebaceous gland and dermomuscular layer of normal mouse skin.2. M3R is dectected in PMNs, MNCs and FBCs during the skin incised wound healing in mice.3. The time-dependent expression of M3R may be used as a marker for the wound age determination of the incised skin. |
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