Morphometric Study of Skin Vessels After Mechanical Injury Based on von Willebrand Factor Antibody Labeling

Abstract

BACKGROUND: Angiogenesis is one of the most important factors of histogenesis in tissue regeneration. In the evaluation of wounds of various etiologies, identifying blood vessels in hematoxylin- and eosin-stained specimens is often challenging. This difficulty arises from the histological structure of the vascular wall and the complex histotopographic arrangement of vessels. Immunohistochemical staining with antibodies to von Willebrand factor, a specific endothelial cell protein, is one of the most informative methods for vascular detection. Applying this technique to the study of wound healing enhances the assessment of the histotopography and morphology of the microvascular network involved in skin repair.

AIM: To assess the changes in the number and size of skin vessels at various stages of regenerative histogenesis following mechanical injury, using immunohistochemical staining with antibodies against von Willebrand factor.

METHODS: A single-center, controlled, randomized, nonblinded experimental study was conducted. The study material consisted of skin samples from the mid-thigh region of Wistar rats collected at different time points during wound healing after a deep incised injury. The animals were divided into 9 groups: the control group (n = 3) included intact animals, whereas the remaining groups (3 animals per group) corresponded to different time points of removement from the experiment — 12 hours, 24 hours, and 2, 3, 6, 10, 15, and 25 days after injury. At each time point, skin biopsies were processed for histological examination with immunohistochemical staining using antibodies to von Willebrand factor, followed by morphometric analysis of the resulting digital images.

RESULTS: Blood vessels were visualized in the dermis and hypodermis of rat skin and classified into 4 groups according to their caliber (visible cross-sectional area). The most pronounced changes were observed in small-caliber vessels (cross-sectional area ≤ 100 μm²). These vessels were absent in intact skin specimens but appeared in the experimental groups from day 2 through day 10 after injury. In skin samples obtained on days 15 and 25 after injury, a gradual decrease in the number of vessels with a cross-sectional area ≤ 100 μm² was noted. Similar trends were observed for vessels of medium (100–500 μm²) and large (500–1000 μm²) caliber. Vessels with a cross-sectional area ≥ 1000 μm² were rare, and their number did not correlate with the wound healing phase.

CONCLUSION: Immunohistochemical staining with anti–von Willebrand factor antibodies in rat skin sections demonstrated good reproducibility and yielded high-quality specimens. In the experimental wound healing model, the method exhibited high selectivity in identifying blood vessels. Morphometric analysis of histological samples confirmed a correlation between vessel count and the sequential phases of the wound healing process.

Keywords

skin / dermis / hypodermis / wound healing / immunohistochemistry / von Willebrand factor / blood vessels

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