Abstract
In forensic practice, it is important to diagnose wound age accurately. We analyzed the proteome of injured murine skin to identify a novel protein marker of wound age after recent injury. We used samples from 3days after injury, with 0days as the control. The proteins were separated with two-dimensional electrophoresis. Using mass spectrometry, we identified a protein, chitinase-like 3 (Chil3). Chil3 messenger RNA (mRNA) expression showed temporal changes, which included a peak increase at 2days after injury. Next, we produced an anti-Chil3 antibody and confirmed its specificity with western blotting. Similar to the mRNA results, an analysis of temporal changes in Chil3 protein expression revealed a peak at 2days after injury. We also investigated the time course of changes in Chil3 tissue localization using immunohistochemistry. Chil3 signals remained in the wounded area for up to 9days. However, Chil3-positive cells were observed in the scab, the edge of the dermal layer, and neogenetic granulation tissue between 1 and 3days. Thus, wound age can be histologically determined using the localization of Chil3 but not its general existence. Additionally, double-labeled fluorescent immunohistochemistry revealed that the Chil3-expressing cells were mainly neutrophils. These data show that Chil3 is expressed in neutrophils during the early stage of wound healing in mice; thus, Chil3 is a potential histological marker of 1-3-day-old wounds.
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