Histological and miRNAs postmortem changes in incisional wound

Wound has been defined as a disruption of the continuity of a tissue structure due to application of mechanical force (Saukko Pekka 2004).

Incisional wounds have been and will remain a major cause of violent death. The nature of the wound, as ante-mortem or postmortem, have to be ascertained by the forensic pathologist (Karlsson 1998; Siddiqui et al. 2016).

Determination of wound vitality in perimortal injuries, and the interpretation of the chronological sequence of events is an important part in forensic work (Obac et al. 2011).

Ante-mortem wound shows tissue reaction and consists essentially of an acute inflammation. Accordingly, many biochemical molecules (e.g., miRNAs) and cells (e.g., neutrophil and mononuclear cell) are involved in this inflammatory process (Vinay et al. 2017).

MicroRNAs (miRNAs) are small 21- to 25-bp non-coding single-stranded RNA molecules. They play a critical role in all biological processes (Carthew and Sontheimer 2009). MiR-205 is highly expressed in normal skin, while miRNA-21 is induced after skin injury and promotes wound healing (Long et al. 2018; Yi et al. 2008).

Nature of the wound could be estimated via many gross and microscopic changes. Because of the uncertain and variable results of the gross examination, it is important to study the wound using histological (Vinay et al. 2017) and bio-molecular techniques, e.g., miRNA (Da Silva et al. 2016).

Hence, we aimed to evaluate the effect of postmortem interval on cutaneous incisional wound using histological examination and miRNA-205 and miRNA-21 expressions.

Prior to conducting this experiment, the study protocol was approved by the Ethical Committee of King Saud University, the institutional animal care and use committee, King Saud University.

This study was conducted on 18 female albino rats with incisional wound.

Trauma is the leading cause of mortality all over the world (Eming et al. 2014). Different histological and molecular changes occur in the incisional wound (Paladini et al. 1996). Forensic scientists could use these changes to evaluate the effect of the time passed since death in the incisional wounds (Gunn et al. 2014; Lv et al. 2014).

Few researchers had described the forensic aspect of histologic changes of the skin injuries (Siddiqui et al. 2016; Obac et al. 2011; Vinay et al. 2017; Vidinský et al. 2006). Most of these studies were conducted on blunt injuries. No such study was conducted to detect molecular and histological changes in incisional injuries occurred just before death. Therefore, there is a need for such study.

Skin-wound healing starts immediately after mechanical damage. It consists of three phases: inflammation, proliferation, and maturation. Following trauma, there is an overlap in the early inflammation phase and can be recognized as healing signs of wound vitality (Obac et al. 2011).

Vidinský et al. (Vidinský et al. 2006) studied the rats’ skin morphology for a period of 7 days after incisional wound. During 24 h after incision, they noted intense acute inflammatory reaction in the wound area in the form of cellular infiltration, the thickened epidermis at wound margins, and fibrin network filling the incisional space. Starting from day 2 to 7, they noted healing process in the form of a scab formation, thickened epidermis, keratinocyte migration, starting re-epithelization, and randomly distributed fibroblasts with no collagen formation.

Similarly, in the our specimens, acute inflammatory cells appeared at wound margin immediately after incision, and with increasing time passed since injury infliction, incisional wounds showed collagen, fibrous, and hemosiderin depositions.

Obac et al. (Obac et al. 2011) evaluated fibrin deposition; a marker of vital reaction, in rats’ skin after incisional injuries. The fibrin accumulated at the margins of the ante-mortem incisions (1 h after incision). They concluded that fibrin is feasible to distinguish between ante-mortem and postmortem lesions.

The incisional injury in our study showed significantly higher miRNA-205 and miRNA-21 expressions in group II (24 h after death) than other groups. Although there is a rapid decrease in miRNA-205 and miRNA-21 expressions in wounded samples, this decrease did not show any statistical significant correlation with postmortem interval. Of note, short duration passed after death did not show a significant increase in mRNA degradation.

A previous study had concluded that miR-205 expression significantly increased in the wound area at 12 h after wound infliction. Overexpressed miR-205 accelerated the cellular regrowth rate, cellular migration, and cellular proliferation during wound-healing process (Lin et al. 2013).

Long et al. (Long et al. 2018) studied the effect of miR-21 on wound healing process in aged mice. They found that miR-21 expression retained a high level in the wound area and allowed better skin repairing capacity. Its level was upregulated at day 7 more than twofold compared to those of day 3. Its dysregulation may contribute to impaired wound repair.

Ludwig et al. (Ludwig et al. 2016) reported that the miR-205 was highly expressed in human skin and can resist environmental variations and PM degradation. This trend could be related to its short length 21–25 bp. Moreover, McClure et al. (McClure et al. 2014) stated that genetic susceptibility and health condition could affect miR-21expression.

Molecular degradation in dead bodies increases, as the postmortem duration increases. Therefore, gene expressions decrease constantly with increasing time after death (Bauer et al. 2003; Sampaio-Silva et al. 2013).

The studied miRNA had been continuously expressed up to 48 h after death, but at lower level. MicroRNAs can resist ribonuclease degradation due to their being tightly bound to the active RNA-induced silencing complex and play special functions in keeping alive cells after death. Hence, miRNAs can be continuously expressed (Wang et al. 2016).

In this study, we had not demonstrate a significant negative correlation between miRNA expressions and time passed since death. Our results were inconsistent with other studies (Lv et al. 2014; Bauer et al. 2003; Sampaio-Silva et al. 2013; Wang et al. 2013). Possible reasons for these conflicting results could be related to studied molecular molecule, examined tissue, and chosen postmortem duration (Ali et al. 2017).

Our findings were augmented by the Nagy et al.’s findings (Nagy et al. 2015), who also demonstrated that miRNA expressions in postmortem brain tissue were stable and continuously expressed for up to 4 days.

When the results of the present study were compared with published literature, the histologic pattern and bio-molecular findings are more or less similar, but the progression of histologic and bio-molecular changes were different. The reason for this difference is related mainly to the methodological variations; especially the assessment time. The skin samples in our study were examined after death as postmortem samples of perimortal wound. Moreover, the condition of storage environment can greatly affect the speed of bio-molecular and histological changes (Ibrahim 2018).

Forensic pathologists commonly examine postmortem samples in their practice. Histological examination and reverse transcription polymerase chain reaction are useful techniques to examine these samples.

We found evidence that histological signs of acute inflammation can be detected early in perimortal incisional wounds, starting from time of infliction up to 2 days after death, and can be used as an indicator of wound vitality. However, molecular changes became more evident in determining time passed since death especially at 24 h duration. Although these histological and molecular findings were encouraging, our study had some limitations; for instance, our attempt in analyzing and correlating the histologic changes had been made in a wide time gap.

More researches are needed into histo- and bio-molecular changes of the perimortal wounds within successive shorter intervals from different body sites to augment preliminary results of this study.