Detection of satellite cells during skeletal muscle wound healing in rats: time-dependent expressions of Pax7 and MyoD in relation to wound age.

The study was focused on time-dependent expressions of paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) during skeletal muscle wound healing. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17, and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. By morphometric analysis, the data based on the number of Pax7(+)/MyoD(-), Pax7(+)/MyoD(+), and Pax7(-)/MyoD(+) cells were highly correlated with the wound age. Pax7 and MyoD expressions were upregulated after injury by Western blot and quantitative real-time PCR assays. The relative quantity of Pax7 protein peaked at 5 days after injury, which was >1.13, and decreased thereafter. Similarly, the relative quantity of MyoD mRNA expression peaked at 3 days after injury, which was >2.59. The relative quantity of Pax7 protein >0.73 or mRNA expression >2.38 or the relative quantity of MyoD protein >1.33 suggested a wound age of 3 to 7 days. The relative quantity of MyoD mRNA expression >2.02 suggested a wound age of 1 to 7 days post-injury. In conclusion, the expressions of Pax7 and MyoD are upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting that Pax7 and MyoD may be potential markers for wound age estimation in skeletal muscle.

[Effects of PI3K/Akt Pathway in Wound Healing Process of Mice Skin].

OBJECTIVE: To investigate the expressions and time-dependent changes of phosphatidylinositol-3-kinase (PI3K), phospho-PI3K (p-PI3K), protein kinase B (PKB/Akt) and phospho-Akt (p-Akt) during wound healing process of mice skin. METHODS: The changes of PI3K, p-PI3K, Akt and p-Akt expression in skin wound were detected by immunohistochemistry, Western blotting and real-time PCR. RESULTS: Immunohistochemistry showed the expression of PI3K and p-Akt were observed in mononuclear and fibroblast after skin wound, and reached peak in reconstruction. The positive bands of PI3K, p-PI3K, Akt and p-Akt were observed in all time points of the wound healing process by Western blotting. The expression peak of p-PI3K and p-Akt showed in inflammation and proliferation; the expression peak of PI3K and Akt in reconstruction. Real-time PCR showed the expression peak of PI3K mRNA in inflammation and reconstruction and the peak of Akt mRNA in reconstruction. CONCLUSION: During the wound healing process, the expressions of PI3K, Akt, p-PI3K and p-Akt show different changes with significant correlation to wound time. The expression of PI3K/Akt may be a valuable marker for wound time estimation.

[Errors in six degree-of-freedom pose estimation of spine tumors assessed by image guided radiotherapy].

OBJECTIVE: To evaluate the six-degree setup errors of tumors of cervical vertebra, thoracic vertebra and lumbar vertebra by image guided radiotherapy. METHODS: From May 2013 to June 2014, 30 patients with spinal malignant tumors(10 patients of cervical vertebra, thoracic vertebra and lumbar vertebra respectively) were treated with Elekata Synergy accelerator (Elekta company,Sweden). Six-degree set up errors were corrected using HexaPODTMevoRT bed under image of on board cone beam computed tomography (CBCT) guided. All the patients received kilovoltage CBCT before receiving radiotherapy and after correction. The acquired images were co-registered with planning CT with bone window. The data of 838 CT images were analyzed and the errors of translational directions X(lateral), Y(lngitudinal),Z(vertical)and rotational directions RX(pitch), RY(roll), RZ(yaw) were recorded. The data were compared by t-test using SPSS 13.0. RESULTS: The absolute translational setup errors in X, Y and Z axes of cervical vertebra before correction were (1.71 ± 0.10) mm, (1.81 ± 0.11) mm and (1.94 ± 0.09) mm respectively: (3.17 ± 0.19) mm, (4.26 ± 0.28) mm and (2.18 ± 0.12) mm for thoracic vertebra, and (2.69 ± 0.24) mm, (3.33 ± 0.26) mm and (2.86 ± 0.21) mm for lumbar vertebra. The residual setup errors in X, Y and Z axes of cervical vertebra were (0.5 ± 2.4) mm,(0.01 ± 2.4) mm and (2.4 ± 1.4) mm, respectively after correction;(1.17 ± 0.11) mm,(0.26 ± 0.30) mm and (0.08 ± 0.12) mm for thoracic vertebra and (1.09 ± 0.24) mm,(2.03 ± 1.26) mm and (0.06 ± 0.51) mm for lumbar vertebra. The t-test of paired data of set up errors before and after CBCT showed significant difference in three translational directions of cervical vertebra and thoracic vertebra, only Z(t=-3.518,P<0.001) for lumbar vertebra. The absolute rotational setup errors in RX,RY and RZ axes of cervical vertebra before correction were 0.67° ± 0.04°,1.06° ± 0.06° and 0.78° ± 0.05° respectively. 0.62° ± 0.05°, 0.75° ± 0.06°, and 0.84° ± 0.06° for thoracic vertebra, 0.59° ± 0.06°, 0.80° ± 0.07°, and 0.73° ± 0.06° for lumbar vertebra. The rotational directions RX, RY and RZ axes of cervical vertebra were 0.27° ± 0.14°, 1.20° ± 0.04° and 0.28° ± 0.05° respectively; 0.02° ± 0.20°, 0.05°±0.26° and 0.64° ± 0.16° for thoracic vertebra and 0.09° ± 0.26°, 0.50°±0.05°,and 0.03°±0.16° for lumbar vertebra. The t-test of paired data of set up errors before and after CBCT showed significant difference in three rotational directions of cervical vertebra and lumbar vertebra, only RY(t=7.106, P<0.001)for thoracic vertebra. All the patients acquired pain relief and there was no radiation-induced toxicity detected clinically during a median follow-up of 6 months. CONCLUSION: Six-degree set up errors of spine tumors were corrected effectively with HexaPODTMevoRT bed under CBCT image guided and its feasibility in day-to-day clinical practice has been demonstrated.

The distribution and time-dependent expression of HIPK2 during the repair of contused skeletal muscle in mice.

HIPK2 is an evolutionarily conserved serine/threonine kinase and is considered a co-regulator of an increasing number of transcription factors modulating a variety of cellular processes, including inflammation, proliferation and fibrosis. Skeletal muscle injuries repair is an overlapping event between inflammation and tissue repair. There are no reports about HIPK2 expression in skeletal muscles after trauma. A foundational study on distribution and time-dependent expression of HIPK2 was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR, which is expected to obtain a preliminary insight into the functions of HIPK2 during the repair of contused skeletal muscle in mice. An animal model of skeletal muscle contusion was established in 50 C57B6/L male mice. Samples were taken at 1, 3, 5, 7, 9, 14, 17, 21 and 28 days after contusion, respectively (5 mice at each posttraumatic interval). 5 mice were employed as control. No HIPK2-positive staining was detected in uninjured skeletal muscle. Intensive immunoreactivties of HIPK2 were observed in polymorphonuclear cells, round-shaped mononuclear cells, regenerated multinucleated myotubes and spindle-shaped fibroblastic cells in the contused tissue. The HIPK2-positive cells were identified as neutrophils, macrophages and myofibroblasts by double immunofluorescent procedure. HIPK2 protein and mRNA expression were remarkably up-regulated after contusion by Western blotting and qPCR analysis. The results demonstrated that the expression of HIPK2 is distributed in certain cell types and is time-dependently expressed in skeletal muscle after contusion, which suggested that HIPK2 may participate in the whole process of skeletal muscle wound healing, including inflammatory response, muscle regeneration and fibrogenesis.

[Analysis of features of 15 successful positional cloning of QTL in rice].

As the most efficient strategy in gene clone, positional cloning has been used widely in QTL cloning in rice. The objective of this paper is to make summary features of QTL positional cloning based on 15 successful positional cloning attempts. (1) most of the populations used in the analysis were derived from interspecific or intersubspecies; (2) the target QTL had been identified with very large phenotypic effects; (3) the candidate genomic region was usually narrowed down to 40 kb; (4) the primary mapping result was exact; and the fine mapping population was more than 6,000, while the number of recessive population was more than 1,500. Otherwise, the nodus of QTL positional cloning and the corresponding solving methods were discussed.

α7-nAChR Activation Has an Opposite Effect on Healing of Covered and Uncovered Wounds.

The α7 nicotinic acetylcholine receptor (α7-nAChR) is associated with inflammation, re-epithelialization, and angiogenesis in wound healing process. A recent study demonstrated that PNU-282987, a selective agonist of α7-nAChR, accelerates the repair of diabetic excisional wounds. Whether α7-nAChR activation promotes non-diabetic wounds healing is unknown. The aim of this study was to evaluate the effects of α7-nAChR activation on non-diabetic wound healing. The effects were evaluated in two wound models. In the first model, the wound was covered with a semi-permeable transparent dressing. In the second model, the wound was left uncovered. In both models, the mice were randomly assigned to two treatment groups: saline or PNU282987 (25 mice in each group). In covered wounds, we found that α7-nAChR activation inhibited re-epithelialization, angiogenesis, and epithelial cells proliferation, promoted neo-epithelial detachment, and suppressed neutrophil infiltration and the expression of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). However, in uncovered wounds, we observed that α7-nAChR activation promoted re-epithelialization and angiogenesis, inhibited neutrophil infiltration and the expression of high mobility group box (HMGB)-1, epidermal growth factor (EGF), and VEGF. In conclusion, this data demonstrated that α7-nAChR activation inhibited wound healing in covered wounds but played an opposite role in uncovered wounds. The opposite effect might be primarily due to inhibition of inflammation.

Cannabinoid 2 receptor attenuates inflammation during skin wound healing by inhibiting M1 macrophages rather than activating M2 macrophages.

BACKGROUND: The anti-inflammatory properties of the cannabinoid 2 receptor (CB2R) in injury and inflammatory diseases have been widely substantiated. Specifically, the anti-inflammatory effect of CB2R may be achieved by regulating macrophage polarisation. Several research findings suggested that the activation of CB2R could attenuate inflammation by reducing pro-inflammatory M1 macrophage polarisation and promoting anti-inflammatory M2 polarisation. However, considering CB2R inhibits fibrosis and M2 promotes fibrosis, that the activation of CB2R may lead to an increase in M2 macrophages seems contradictory. Therefore, we hypothesised that the activation of CB2R to attenuate inflammation is not achieved by up-regulating M2 macrophages. METHODS: We established an incised wound model using mouse skin and used this to evaluate the effect of CB2R agonists (JWH133 or GP1a) and an antagonist (AM630) on wound healing. At various post-injury intervals, we used western blot analysis, immunofluorescence staining, enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction assays to determine CB2R protein expression, M1/M2 macrophage infiltration, and the protein and gene expression of M1/M2-associated markers and cytokines in skin lesions. RESULTS: Activation of CB2R significantly reduced M1 macrophage infiltration and slightly increased M2 macrophage infiltration. Similarly, gene expression and protein levels of M1-associated markers and cytokines (interleukin [IL]-6, IL-12, CD86 and inducible nitric oxide synthase) were significantly down-regulated after CB2R agonist administration; in contrast, markers and cytokines were increased in the CB2R antagonist-treated group. Conversely, the administration of agonists slightly increased gene expression and protein levels of M2-associated markers and cytokines (IL-4, IL-10, CD206 and arginase-1 [Arg-1]); however, a statistical significance at most time points post-injury was not noted. CONCLUSION: In summary, our findings suggested that during incised skin wound healing in mice, increased levels of CB2R may affect inflammation by regulating M1 rather than M2 macrophage subtype polarisation. These results offer a novel understanding of the molecular mechanisms involved in the inhibition of inflammation by CBR2 that may lead to new treatments for cutaneous inflammation.

Cannabinoid CB2 receptors are involved in the regulation of fibrogenesis during skin wound repair in mice.

Studies have shown that cannabinoid CB2 receptors are involved in wound repair, however, its physiological roles in fibrogenesis remain to be elucidated. In the present study, the capacity of cannabinoid CB2 receptors in the regulation of skin fibrogenesis during skin wound healing was investigated. To assess the function of cannabinoid CB2 receptors, skin excisional BALB/c mice were treated with either the cannabinoid CB2 receptor selective agonist, GP1a, or antagonist, AM630. Skin fibrosis was assessed by histological analysis and profibrotic cytokines were determined by immunohistochemistry, immunofluorescence staining, reverse transcription­quantitative polymerase chain reaction and immunoblotting in these animals. GP1a decreased collagen deposition, reduced the levels of transforming growth factor (TGF)­ß1, TGF­ß receptor I (TßRI) and phosphorylated small mothers against decapentaplegic homolog 3 (P­Smad3), but elevated the expression of its inhibitor, Smad7. By contrast, AM630 increased collagen deposition and the expression levels of TGF­ß1, TßRI and P­Smad3. These results indicated that cannabinoid CB2 receptors modulate fibrogenesis and the TGF­ß/Smad profibrotic signaling pathway during skin wound repair in the mouse.

Pharmacological activation of cannabinoid 2 receptor attenuates inflammation, fibrogenesis, and promotes re-epithelialization during skin wound healing.

Previous studies showed that cannabinoid 2 (CB2) receptor is expressed in multiple effector cells during skin wound healing. Meanwhile, its functional involvement in inflammation, fibrosis, and cell proliferation in other organs and skin diseases implied CB2 receptor might also regulate skin wound healing. To verify this hypothesis, mice excisional wounds were created and treated with highly selective CB2 receptor agonist GP1a (1-(2,4-dichlorophenyl)-6-methyl- N-piperidin-1-yl-4H-indeno[1,2-c]pyrazole-3-carboxamide) and antagonist AM630 ([6-iodo-2- methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone) respectively. The inflammatory infiltration, cytokine expression, fibrogenesis, and wound re-epithelialization were analyzed. After CB2 receptor activation, neutrophil and macrophage infiltrations were reduced, and expressions of monocyte chemotactic protein (MCP)-1, stromal cell-derived factor (SDF)-1, Interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF)-A were decreased. Keratinocyte proliferation and migration were enhanced. Wound re-epithelialization was accelerated. Fibroblast accumulation and fibroblast-to-myofibroblast transformation were attenuated, and expression of pro-collagen I was decreased. Furthermore, HaCaT cells in vitro were treated with GP1a or AM630, which revealed that CB2 receptor activation promoted keratinocyte migration by inducing the epithelial to mesenchymal transition. These results, taken together, indicate that activating CB2 receptor could ameliorate wound healing by reducing inflammation, accelerating re-epithelialization, and attenuating scar formation. Thus, CB2 receptor agonist might be a novel perspective for skin wound therapy.

A fundamental study on the dynamics of multiple biomarkers in mouse excisional wounds for wound age estimation.

Wound age estimation is a classic but still modern theme in forensic practice. More experiments on different types of wound are needed to further improve its accuracy. In this study, mouse skin excisional wounds were created to simulate dermal defective injury. The neutrophil and macrophage infiltration, fibroblast and fibrocyte accumulation as well as their myofibroblastic transformation were examined. In addition, some wound healing-related molecules, including IL-1ß, IL-6, TNF-α, IFN-γ, MCP-1, CXCL12, VEGF-A, EGF, KGF, pro-col Iα2 and pro-col IIIα1, were quantified by Western blotting and real-time quantitative PCR. Neutrophils and macrophages profoundly infiltrated in the wound at 12 h-1 d and 3 d-10 d respectively. Fibroblasts and fibrocytes accumulated in the wound from 3 d, and transformed into contractile myofibroblasts from 5 d post injury. The transformation ratios of fibroblasts and fibrocytes were highest at 7 d-10 d and 10 d respectively (over 50%). MCP-1 and CXCL12 increased from 12 h to 5 d, and IL-1ß, TNF-α and pro-col IIIα1 up to 7 d. IL-6 and VEGF-A increased from 12 h to 1 d-10 d. Pro-col Iα2 increased from 7 d to 21 d. IFN-γ decreased from 12 h to 10 d. By comprehensive analysis of these factors from the perspective of morphometrics, protein and gene expressions, this study provided us with fundamental information for wound age estimation, especially in the wounds with full-thickness defection.

The distribution and time-dependent expression of MAGL during skeletal muscle wound healing in rats.

Monoacylglycerol lipase (MAGL) is widely distributed in mammals and largely responsible for metabolizing 2-arachidonoylglycerol (2-AG). Little is known about its expression in skeletal muscles after trauma. A preliminary study on time-dependent expression and distribution of MAGL was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR (qPCR) during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17 and 21 days after contusion, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. Weak immunoreactivity of MAGL was observed in the sarcoplasm of myofibers in control rats. Intensive immunoreactivities of MAGL were observed in polymorphonuclear cells (PMNs), round-shaped mononuclear cells (MNCs), spindle-shaped fibroblastic cells (FBCs) and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as MAGL-positive cells by double immunofluorescent procedure. MAGL expression was remarkably up-regulated after contusion by qPCR and Western blot analysis. The results demonstrate that the expression of MAGL is distributed in certain cell types and time-dependently expressed in skeletal muscles after trauma, suggesting that MAGL may be involved in inflammatory response, fibrogenesis and muscle regeneration during skeletal muscle wound healing.

CB2R orchestrates fibrogenesis through regulation of inflammatory response during the repair of skeletal muscle contusion.

Skeletal muscle injuries repair typically is an overlapping event between inflammation and tissue repair. Our previous study has demonstrated that activation of cannabinoid receptor type 2 (CB2R) by JWH-133 alleviates fibrosis in the repair of rat skeletal muscle contusion. Meanwhile, accumulated data show that CB2R stimulation exerts anti-inflammatory property in sepsis and cystitis. However, the effects of CB2R on inflammatory cytokines in response to the repair of skeletal muscle contusion are still unknown. In this study, we used selective agonist or antagonist of CB2R to observe the role of CB2R on inflammation and fibrogenesis during the repair of contused skeletal muscles in rats. Our results revealed that treatment with Gp1a, a selective CB2R agonist, significantly decreased the infiltration of neutrophils and macrophages, the expression of pro-inflammatory cytokines MCP-1, TNF-α, IL-1ß and IL-6, the expression of pro-fibrotic cytokines IL-4, IL-13, TGF-ß and P-Smad3 while increased anti-fibrotic cytokine IL-10 production as compared with Vehicle. The opposite results were observed in the CB2R inhibition group with AM630. Our study demonstrated that CB2R orchestrates fibrogenesis through regulation of inflammatory response during the repair of skeletal muscle contusion.

α7nAChR is expressed in satellite cells at different myogenic status during skeletal muscle wound healing in rats.

Recent study has reported that α7 nicotine acetylcholine receptor (α7nAChR) is expressed in regenerated multinucleated myotubes. But the distribution of α7nAChR in satellite cells in different myogenic status is unknown. A preliminary study on the dynamic distribution of α7nAChR in satellite cells was performed by double indirect immunofluorescent procedures during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17 and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. In normal muscle specimens, weak immunoreactivity for α7nAChR was detected in a few satellite cells (considered as quiescent). α7nAChR-positive signals were observed in proliferated and differentiated satellite cells and regenerated multinucleated myotubes in the wounded areas. By morphometric analysis, the average number of α7nAChR+/Pax7+ and α7nAChR+/MyoD+ cells climaxed at 5 days post-injury. The average number of α7nAChR+/myogenin+ cells was significantly increased from 3 to 9 days post-injury as compared with other posttraumatic intervals. The protein level of α7nAChR maximized at 9 days post-injury, which implies that α7nAChR was associated with the satellite cells status. Our observations on expression of α7nAChR in satellite cells from quiescence to myotube formation suggest that α7nAChR may be involved in muscle regeneration by regulating satellite cell status.

The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats.

Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and pro-inflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB1 and CB2 receptors.