Angiogenesis assessment in experimental third degree skin burns: a histological and immunohistochemical study

In the past 30 years, after the discovery of vascular proliferation factors, angiogenesis is one of the most intensively studied fields. Restoring the vascular network after burn injury is essential for healing, as it brings oxygen and nutrients to injured tissues, improves the contribution of inflammatory cells and prepares the damaged area for repair and tissue regeneration. To study the process of angiogenesis we used seven groups of five animals, each of adult Wistar rats, which were inflicted with third degree skin burns. From each group of animals, we sampled at successive intervals of three days the entire burnt wound with a ring of surrounding normal skin. Sampled skin fragments were processed for paraffin inclusion, sectioned with a microtome and stained with Hematoxylin-Eosin or Masson trichrome. The samples were also analyzed using single chromogenic immunohistochemistry or double immunofluorescence for the presence of CD34 and alpha smooth muscle actin (α-SMA). Angiogenesis process started at about three days after the burn infliction, with the appearance of tubular structures lined by CD34-positive cells. Subsequently, these cells showed intense proliferative activity that generated a network that included progressive neovascularization around the wound surface. Maximum vascular proliferation occurred at 9-15 days after injury, when the number of capillaries reached 229/mm², and the total area of capillary angiogenesis at 100.27 μm² (about 10% of the section area). Subsequently, the process of angiogenesis was gradually reduced, but remained at moderate levels after wound healing. During the process of angiogenesis, there was a very close relationship between CD34-positive cells and pericytes (as α-SMA-positive).