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Wound healing evaluation is important in forensic pathology, in which angiogenesis plays an important role. We have already shown that vascular endothelial growth factor A (VEGF) is produced in the rat skin incision wounds by neutrophils, endothelial cells, and fibroblasts. In this study, we assessed the changes in the mRNA expressions of various factors possibly involved in angiogenesis including angiopoietin (ANGPT) 1 and 2, cadherin 5 (CDH5), granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF), granulocyte colony stimulating factor (CSF3/G-CSF), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand12 (CXCL12/SDF1), endothelin 1 (ET1), fibroblast growth factor 1 (FGF 1), hepatocyte growth factor (HGF), hypoxia inducible factor 1 alpha (HIF1a), leptin, matrix metallopepitidase 9 (MMP9), serpine/plasminogen activator inhibitor1 (PAI1), platelet-derived growth factor-A (PDGF-A), transforming growth factor alpha and beta 1 (TGFa and b1), tenomodulin (TNMD), and troponin I type 2 (TNNI2) in the early stage of the rat skin incision wounds by real time RT-PCR. Factors reported to be involved in lymphangiogenesis such as fibroblast growth factor 2 (FGF 2), c-fos induced growth factor (FIGF/VEGF-D), forkhead box C2 (FOXC2), and prospero homeobox 1 (PROX1) were also studied. One and 3 days after the dorsal skin incisions, wounds on male Sprague-Dawley rats showed the statistically significant increases in the mRNA expressions for CXCL2, CSF3, MMP9, PAI1, and CSF2, whereas TGFa, TNNI2, FGF1, TNMD, leptin, and CXCL12 showed the statistically significant decreases. Interestingly, lymphgangiogenic factors FOXC2, PROX1, and FGF2 also showed the statistically significant decreases. In situ hybridization and immunohistochemistry showed the mRNA and protein positivity in endothelial cells, fibroblasts, and some leukocytes at the bottom of the wound tissue for PAI1, CSF3, and MMP9, 1 day after the skin incisions. Our novel findings show the possible involvement of several factors involved in angiogenesis and lymphangiogenesis in the early stage of wound healing process, which may be useful for forensic wound evaluations., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Wound healing evaluation is important in forensic pathology. We have already shown that vascular endothelial growth factor (VEGF) is produced in the rat skin incision wounds. In this study, we used cyclophosphamide hydrate (CPM) to induce leukocytopenia in rats, and measured VEGF in the skin incision wound area to assess the involvement of leukocytes in the early production of VEGF. Male Sprague-Dawley (SD) rats were intraperitoneally administered CPM 75 mg/kg body weight on day 0 and 5, and dorsal skin incision wounds were made on day 5. One and 3 days after the skin incision, leukocytes counts were determined and skin specimens from the wounds were collected for measurements of total proteins, VEGF proteins, and semi-quantification of VEGF mRNA. VEGF immunohistochemistry and in situ hybridization for VEGF mRNA were also performed. VEGF proteins were smaller in the amount statistically significantly in the 1- and 3-day-old wounds of CPM-induced leukocytopenic rats, whereas VEGF mRNA was increased compared with saline-treated control rats. Immunohistochemically, VEGF was positive in leukocytes and mesenchymal cells including fibroblasts and endothelial cells in the 3-day-old wound of saline-administered control rats, while a few fibroblasts and endothelial cells were positively stained in CPM-administered rats. In situ hybridization showed the localization of VEGF mRNA in mesenchymal cells including fibroblasts and endothelial cells in the 1-day-old wound of CPM-administered rats, whereas saline-administered control rats also showed VEGF mRNA positivity in leukocytes. Our study indicates that leukocytes may be the major source of VEGF in the early stage of the rat skin incision wound., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

In the wound healing process, angiogenesis is involved in the recovery of vasculature, and its process has been investigated. On the other hand, the reconstruction of lymphatic vessels in the injured subcutaneous tissue has not been studied in detail. We studied the recovery of lymphatic vessels using podoplanin immunohistochemistry in the paraffine section microscopy of the rat skin incision wound. Our result indicates a novel finding that subcutaneous tissue of the incised skin area does not show any recovery of lymphatic vessels up to 84 days after the skin incision. As the wound area shrunk, the surrounding subcutaneous tissue covered with the normal skin epithelial cells approached toward the center of the wound, and the lymphatic vessels in the surrounding tissue gradually reached the incision wound area. On the other hand, the regeneration of the vasculature occurred within the wound area as assessed by CD31 and von Willebrand Factor (vWF) immunohistochemistry. This difference was confirmed by the morphometric quantification of podoplanin- or vWF-positive vessels. Our results show that there is a clear difference in the recovery pattern of vascular and lymphatic vessels in the skin wound healing process.