Proliferation to apoptosis ratio in cutaneous connective tissue cells during mechanical injury healing in an experiment

BACKGROUND: The proliferation to apoptosis ratio at a wound site is crucial for healing. Regenerative histogenesis of the skin in the perinecrotic zone is of key interest, specifically the connective tissue layers (the dermis and hypodermis). This zone is characterized by cambial elements of epithelial and connective tissues that drive regeneration, as well as distinctive cell death processes. Immunohistochemical methods are typically used to investigate patterns of histogenetic processes, including proliferation and cell death, in tissues with varying regenerative potential. However, selecting markers that reflect the proliferation to apoptosis ratio at different stages of regeneration remains challenging.

AIM: This study aimed to perform an immunohistochemical evaluation of the proliferation to apoptosis ratio in cutaneous connective tissue cells at different stages of mechanical injury healing.

METHODS: An experimental single-center, continuous, controlled, randomized, non-blind study was conducted. Skin samples of the thigh from Wistar rats were obtained at various stages of healing after mechanical injury (deep incised wound). Animals were divided into nine groups: a control group of intact rats (n = 3) and eight experimental groups corresponding to post-injury time points of 12 hours, 24 hours, 2 days, 3 days, 6 days, 10 days, 15 days, and 25 days (n = 3 per group). Tissue fragments were processed for histological and immunohistochemical examination. Antibodies to phosphorylated histone H3 were used to assess proliferation, whereas apoptosis was detected using antibodies to p53 and caspase-3.

RESULTS: Immunopositive cells expressing phosphorylated histone H3, caspase-3, and p53 were identified in cutaneous connective tissue samples in all experimental groups. The proliferation index was determined, and changes in pro-apoptotic protein expression were analyzed in intact skin and the perinecrotic zone at different stages of regeneration. Based on these data, the proliferation to apoptosis ratio and an index characterizing both processes were calculated. The proliferation to apoptosis ratio was highest when proliferation prevailed over cell death (in intact skin and at the final stages of regeneration) and lowest when apoptosis predominated (inflammation and necrosis phases).

CONCLUSION: For the first time, immunohistochemistry with antibodies to phosphorylated histone H3 was used to investigate regeneration in skin wounds. This marker, which is expressed in proliferating cells, in combination with apoptosis markers, allows assessing the proliferation to apoptosis ratio at different stages of regeneration.