Vascular endothelial growth factor in the early stage of skin incision wounds in cyclophosphamide-induced leukocytopenic rats

Abstract 

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 75mg/kg body weight on day 0 and 5, and dorsal skin incision wounds were made on day 5. One and 3days 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.

Keywords: Vascular endothelial growth factor, Cyclophosphamide, In situ hybridization, Immunohistochemistry, Real-time RT-PCR, Leukocytes