Histopathological, immunohistochemical, and molecular studies for determination of wound age and vitality in rats

Abstract

In forensic medicine, it is vital to verify with the best attainable accuracy once injuries occurred during vital or post-mortem conditions. An immunohistochemical study was carried out to examine the time-dependent expression of macrophage-specific gene CD68 (cluster of differentiation 68), alpha-smooth muscle actin (α-SMA), and vascular endothelial growth factor (VEGF) in different skin wound timings (0, 1, 3, 5, 7, and 14 days) in rats. Histopathological studies were performed to assess the wound age and vitality. Eighteen male albino Wister rats (weighing 170-200 g) were used for wound induction. Rats (n = 3) were euthanised at 0, 1, 3, 5, 7, and 14 days from the starting point of wound induction. Histopathological examination showed that the epidermal re-epithelialisation was completed 14 days after skin incision. The inflammatory phase was recorded during the first 3 days of healing and reached the maximum levels at 5 days, then declined after 7 days, and completely removed at 14 days. The beginning of the proliferative phase was dated to day 3 and the peak at days 5 and 7. The initiation of the granulation tissue formation and remodelling phase of the healing process was observed 5 days after wounding. By immunohistochemical staining, negative VEGF gene expressions at early stages (0-3 days) were observed, as well as neither CD68+ macrophages nor α-SMA+ myofibroblast cells were detected. By increasing the wound ages (5-7 days), granulation tissue and angiogenesis were observed, with the migration of macrophages and fibroblast, which expressed VEGF, CD68, and α-SMA positive reaction. Time-dependent expression of the above markers suggested that they would be useful indicators for the determination of wound age. Both VEGF and transforming growth factor-beta 1 (TGFb1) mRNA levels were determined in different skin wound ages. The transcription of TGFb1 and VEGF increased shortly after wounding, until post-wounding day 7. It then declined constantly, reaching minimal values on day 14.

Keywords: TGFb1; VEGF; gene expression; immunohistochemistry; wound aging.