Estimation of Early Postmortem Interval from Long Noncoding RNA Gene Expression in the Incised Cutaneous Wound: An Experimental Study

Abstract

The assessment of alteration of postmortem RNA expression has forensic significance in estimating postmortem interval. To evaluate wound healing progression and the effect of different postmortem intervals, histopathological changes, immunohistochemical matrix metalloproteinase-9 (MMP-9) expression, and long noncoding fatty acid oxidation (lncFAO), RNA expression was assessed in the incised cutaneous wound model. A full-thickness cutaneous wound was inflicted on 75 rats. All 15 rats were sacrificed at different post-infliction intervals (0, 2, 4, 8 and 10 days), and the cutaneous wounds (n = 5) were excised at different postmortem intervals (0, 5, and 24 h after euthanasia). The maximal inflammatory healing stage was detected at day 4 post-infliction, while near complete healing, thick mature collagen deposition was detected at day 10 post-infliction. LncFAO expression was significantly over-expressed with increasing wound age. MMP-9 was detectable on injury day with continuous elevation until 8 days post-wounding, which later decreased. Although histopathological and immunohistochemical examinations within 24 h postmortem did not show any remarkable changes, lncFAO RNA expression showed a significant negative correlation with hours passed since death. The combined use of histopathological changes, immunohistochemical expression of MMP-9, and molecular expression of lncFAO could be appropriate in wound dating verification. Among these factors, lncFAO could be a reliable indicator in postmortem interval estimation.

Keywords: forensic medicine; incised wound; long non-coding RNA; matrix metalloproteinase-9; postmortem interval.

Figure 1

Photograph showing wound healing progression on day 0, day 2, day 4, day 8, and day 10 post-wounding at different postmortem intervals (0, 5th, and 24th h); each grade in the scale equal 1 mm.

Figure 2

Photomicrograph of wound healing progression (40× magnification) in day 0, day 2, day 4, day 8, and day 10 post-wounding at different postmortem intervals (0, 5th, and 24th hours). Day 0 showed epidermal ulceration (arrow) with mild dermal edema, while on the 2nd day, there were moderate edema, congestion and inflammatory cellular infiltrate with mid fibroplasia (arrow). On the 4th day, wounds showed marked edema, congestion and inflammatory cellular infiltrate with moderate fibroplasia (arrow) and early attempts of re-epithelialization, and on the 8th day, it showed mild to moderate edema, congestion, and inflammatory cellular infiltrate with moderate fibroplasia (arrow) and near complete re-epithelialization. On the 10th day, wounds showed complete re-epithelialization (arrow) with disappearance of inflammatory parameters. No obvious change was detected at different early post-mortem intervals.

Figure 3

Representative of Masson Trichome staining (magnification, ×40) and metalloproteinase-9 expression (magnification, ×400) of cutaneous wound lesions on 0, 2nd, 4th, 8th, and 10th days post-wounding where wound at 0 day showed. Collagen was represented with black arrows, and matrix metalloproteinase-9 was represented with dashed black arrows.

Figure 4

The expression of lncFAO gene in 0, 2, 4, 8, 10 days after wound infliction at different postmortem intervals (0, 5th, and 24th h).

Figure 5

A schematic presentation of macrophage driven acute wound healing. During its three phases; inflammation, proliferation and remodeling. Pro-inflammatory macrophages phagocytose cellular debris and pathogens and release proinflammatory cytokines along with proteolytic enzymes, including metalloproteinases, especially MMP-2, and MMP-9. The macrophage populations begin transitioning to anti-inflammatory pro-wound healing macrophages; this transition is up-regulated by lncFAO that secrete factors, including metalloproteinases, especially MMP-1, MMP-8, MMP-9, that enhance vascular formation, granulation tissue formation, collagen deposition, and re-epithelialization. The anti-inflammatory pro-resolving macrophages enhance granulation tissue breakdown, allow collagen maturation, and improve regenerated skin strength along with down-regulation of a variety of MMPs, including MMP-9. Below the diagram, the main mechanism of lncFAO during the macrophage transition process through activating the β-subunit of mitochondrial trifunctional protein (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase β-subunit; HADHB), a keyenzyme in fatty acid oxidation.