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.

Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice.

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 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.

[Time-dependent recruitment and differentiation of fibrocytes in mouse skin wound healing].

OBJECTIVE: To investigate the time-dependent recruitment and differentiation of fibrocytes in skin wound healing. METHODS: Fibrocytes (expressing CD45 and procollagen I ) and myofibroblasts (expressing CD45 and alpha-SMA) were co-localized by immunofluorescent staining. The number of fibrocytes and myofibroblasts was counted at different post-wounding interval. RESULTS: At 3 d after injury, fibrocytes started to recruit at the margin of the wounds. At 5 d after injury, myofibroblasts started to appear in new formed granulation tissue. The number of fibrocytes and myofibroblasts peaked at 7 d post-wounding. CONCLUSION: During skin wound healing, myofibroblasts in granulation tissue originated at least partly from fibrocytic differentiation. The time-dependent recruitment and differentiation of fibrocytes may provide new information for wound age determination.

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.

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.

[Expression of M3 subtype of muscarinic receptor during the skin incised wound healing in mice].

OBJECTIVE: To investigate the expression of M3 subtype of muscarinic receptors (M3R) during the incised wound healing of the skin in mice and the characteristics of its time-dependent. METHODS: The change of M3R in skin incised wound was detected by immunohistochemical staining and Western blot. RESULTS: M3R-positive cells were detected in epidermis, hair follicle, sebaceous glands, sweat glands, dermomuscular layer in normal mouse skin. Expression of M3R was mainly detectable in polymorphonuclear cells (PMNs) in the wound specimens aged from 6h to 12h after injury. Afterwards, the M3R-positive cells were mostly mononuclear cells (MNCs) and fibroblastic cells (FBCs) at 1 d to 3d post-injury, whereas the M3R-positive cells were mostly FBCs aged from 5 d to 14d. Morphometrically, the ratio of the M3R-positive cells increased aged from 6h to 12h after injury, with a peak at 12h. The ratios kept a high relatively level aged from 1 d to 5 d, but significantly that lowered as compared with aged 12h after injury. The ratio reached the peak at 7 d again after injury, and then decreased gradually. The M3R protein also revealed a time-dependent tendency with double peaks at 12h and 7 d after injury as detected by Western blotting. CONCLUSION: M3R is time-dependently expression in PMNs, MNCs and FBCs suggesting that it may play roles during the skin incised wound healing, and M3R may be used as a marker for wound age determination.

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

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.