The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. 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, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

[Establishment of skin scald model in mice].

OBJECTIVE: To establish the model of skin scald in mice for the study of skin thermal injuries. METHODS: After anaesthetization mice were scalded in a 1 cm-diameter circle area on the central dorsum by boiling water at contact times: 10s or 25s. The mice were sacrificed at 1, 3, 5, 7 and 10 days after scald. The skin samples were collected and analyzed by gross and histopathological examinations. RESULTS: Deep II degree thermal injury involving full-thickness skin was observed in the 10s scald group. III degree thermal injury involving full-thickness skin and the dorsal skeletal muscle was observed in the 25 s scald group. CONCLUSION: A mouse skin scald model is established which is stable and can be used on the skin thermal injury in future research.

[The number of circulating fibrocytes of skeletal muscle in rats after contusion].

OBJECTION: To investigate the time-dependent appearance of circulating fibrocytes of skeletal muscle in rats after contusion. METHODS: The model of skeletal muscle wound was established in rat. The circulating fibrocytes in contused skeletal muscle were detected by CD45 and procollagen I double immunofluorescence staining method. RESULTS: In the control group, CD45- and procollagen I-positive cells were not detected in skeletal muscle. A few CD45 cells were observed aged from 6 h to 1 d after contusion. A few CD45- and procollagen I-positive cells (fibrocytes) initially gathered in injury area 3d after injury. The ratio of positive fibrocytes significantly increased 5 d after injury. The ratio of fibrocytes was highest at 7 d after contusion and then decreased. The volume of fibrocytes showed bigger with injury time increase compared with 3 d group. The expression of procollagen I and CD45 were weakened at 14d after injury. CONCLUSION: The circulating fibrocytes are detected in contused skeletal muscle in time-dependent pattern. Circulating fibrocytes may be a marker in the wound age determination for contused skeletal muscle.

Nicotinic acetylcholine receptor α7 subunit is time-dependently expressed in distinct cell types during skin wound healing in mice.

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.

Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice.

The study investigated the expression of monoacylglycerol lipase (MGL) during the skin-incised wound healing in mice and applicability of the time-dependent expression of MGL to wound age determination by immunofluorescent staining, Western blotting, and real-time PCR. Furthermore, cell types were identified by double immunofluorescence. A total of 45 BALB/c male mice were used in this study. After a 1.5-cm-long incision in the central dorsum skin, mice were killed at intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. In the control, there was a low-level expression of MGL in the epidermis, hair follicles, and glandulae sebaceae. In the injured skin, MGL immunoreactivity was mainly detected in the neutrophils, macrophages, and myofibroblasts. Morphometrically, the average ratios of MGL-positive cells were more than 50% at 5 and 7 days post-wounding, whereas it was <50% at the other posttraumatic intervals. By Western blotting analysis, the average ratio of MGL protein expression was highest at 5 days after injury, which had a ratio of >2.30. Similarly, the relative quantity of MGL mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >2.54. In conclusion, MGL expression is detected in neutrophils, macrophages, and myofibroblasts and significantly up-regulated, suggesting that it may play roles in response to inflammation during skin-incised wound healing. From the viewpoint of forensic pathology, MGL detection is applicable to skin wound age determination.

[Nifedipine attenuates vascular inflammation via inhibin NF-κB activity].

OBJECTIVE: To explore the effects and related mechanism of nifedipine on vascular inflammation induced by cuff placement. METHODS: Adult male C57BL/6J mice (10 to 12 weeks of age) were assigned to control (no cuff placement without nifedipine), cuff placement (cuff placement without nifedipine) and treatment (cuff placement with nifedipine 1 or 5 mg×kg(-1)×d(-1)) groups. Activity of NF-κB in injured artery was measured 5 days after operation. MCP-1 expression and nuclear translocation of NF-κB were examined in injured artery 7 days after operation. RESULTS: DNA-binding activity of NF-κB was significantly increased in the injured artery 5 days after cuff placement which could be downregulated by nifedipine 5 mg×kg(-1)×d(-1). MCP-1 mRNA expression in the injured arteries was increased 7 days after cuff placement and which could be significantly attenuated by nifedipine 5 mg×kg(-1)×d(-1). Cuff placement decreased the cytoplasmic level of p50, IκBα, IκBß, and increased the nuclear level of p50. Nifedipine 5 mg×kg(-1)×d(-1) significantly attenuated these changes. CONCLUSION: Our results suggest that high dose nifedipine could suppresses expression of MCP-1 induced by injured arteries via the inhibin NF-κB DNA binding activity, thereby attenuating vascular inflammation.

Nrf1 is time-dependently expressed and distributed in the distinct cell types after trauma to skeletal muscles in rats.

Our goal was to elucidate the dynamic expression and distribution of the nuclear factor erythroid-derived factor 2-related factor 1 (Nrf1) by immunohistochemistry, Western blotting, and real-time PCR during wound healing of contused skeletal muscle in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male healthy rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. A weak immunoreactivity of Nrf1 was observed in the sarcoplasm and nuclei of normal myofibers in control rats. Prominent immunostaining for Nrf1 was seen in a large number of polymorphonuclear cells, round-shaped mononuclear cells and spindle-shaped fibroblastic cells, and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as expressing Nrf1 by double immunofluorescent procedures. By real-time PCR analysis, Nrf1 expression was up-regulated and peaked at inflammatory phase. The expression tendency was also confirmed by Western blot. In conclusion, Nrf1 is time-dependently expressed in certain cell types, such as neutrophils, macrophages, myofibroblasts and regenerated multinucleated myotubes, suggesting that Nrf1 may modulate oxidative stress response and regeneration after trauma to skeletal muscles.