Introduction: Apoptotic mechanisms are thought to be important in wound healing for the removal of inflammatory cells and evolution of granulation tissue. However, little is understood about the signal, propagation, and mechanisms responsible for triggering cell death in tissue injury, particularly during fetal wound repair. Understanding these signals may lead to insights regarding scarless wound healing. We hypothesized that differences in apoptosis would exist in mid- (E15) compared with late-gestational (E18) mice subjected to cutaneous wounds. We examined early apoptotic signals that may be initiated following tissue injury.
Methods: Pregnant, time-dated mice underwent laparotomy and hysterotomy on embryonic day 15 (E15) and 18 (E18). Full-thickness, excisional cutaneous wounds were made on the dorsum of the fetuses and dorsal skin harvested 15 and 45 min after wounding. Unwounded dorsal skin from additional fetuses collected at the same time points served as controls. The skin was processed to obtain protein, then levels of caspase 3, caspase 7, and poly ADP-ribose polymerase (PARP) were measured by Western blot. Cyclophilin levels were measured to ensure equal loading of protein. Histone-associated DNA complex formation was examined to provide further evidence of cellular apoptosis.
Results: There were no differences in total caspase 3 levels between E15 and E18 fetal tissue with or without wounding, nor was any cleavage of caspase 3 noted in any group. However, cleaved caspase 7 was present in the E15 skin with a >2-fold increase following wounding at both 15 and 45 min, yet absent in the E18 groups. Furthermore, levels of cleaved PARP were also increased by >2-fold at both 15 and 45 min in E15 wound groups, whereas a relatively small amount was only seen in the E18 wound groups at 45 min. DNA-histone fragmentation ELISA assay showed a 5-fold increase in the enrichment of histone-associated DNA fragments in the E15 wounded tissue compared with the time-matched controls at 45 min. This was not seen with the E18 tissue.
Conclusions: Previously, we demonstrated that cutaneous wounds in E15 fetal mice heal in a scarless manner, while similar wounds in E18 mice heal with scar formation. Results from our current work demonstrate differences in apoptosis in mid- compared with late-gestational mouse skin as well as shortly after wounding. Our results suggest that in mid-gestational wounds, activation of apoptotic pathways may be mediated through effector caspase 7 signals with inactivation of PARP. This initiation of apoptotic signals following tissue injury may play a role in scarless wound repair.