Tissue Extract Fluid Cytokine Levels as Markers for Wound Vitality

Objectives: To evaluate ability of estimation of tissue extract fluid (TEF) levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 for differentiation between antemortem (AM) and postmortem (PM) wounds and for rough determination of wound age. Materials & Methods: The study included 20 patients arrived to Surgical Emergency Department (ED) with severe trauma requiring surgical intervention and died after a known survival interval. Time elapsed since trauma inflection till arrival to ED was determined. Four skin biopsies were obtained from each patient: one AM and 3 PM specimens. Skin samples were homogenized and TEF was used for ELISA estimation of IL-1β, TNF-α and IL-6 levels. Results: AM TEF levels of IL-1β and TNF-α were significantly higher compared to PM levels with significantly higher levels in PM1 and PM3 specimens than PM2 specimens. TEF levels of IL-6 were significantly higher in PM2 compared to AM, PM1 and PM3 specimens with significantly higher levels in AM specimens compared to PM3 specimens. TEF levels of IL-1β and TNF-α showed positive significant correlation with wound vitality. TEF levels of IL-6 showed a positive significant correlation with time lapsed since wound inflection. Only elevated TEF level of IL-1β showed significantly high sensitivity for identification of wound inflected since ≤60 minutes AM Conclusion: TEF levels of IL-1β, TNF-α and IL-6 could differentiate between wounds inflected while victim was alive and PM wounds. TEF level of IL-1β works better for such differentiation and could determine wound inflected within 60 minutes with high sensitivity.


Introduction
The term wound describes the morphologicfunctional disruption of the continuity of a tissue structure. A wound can be inflicted during lifewhen the cardiovascular and respiratory system is still intact-or after death, i.e. after cardiac and respiratory arrest. Traumatization during life triggers vital reactions that do not occur in postmortem wound [1].
Three types of vital reactions in wound healing can be distinguished: reactions of the scavenger type, which are almost exclusively mediated by blood cells; reactions by complex signal transduction pathways, which involves cascade-like release of chemokines, cytokines and adhesion molecules and may influence type 1 and type 3 reactions and reactions of the scarring type, which involve the final repair of the damaged tissue and are carried out primarily by cells residing at the wound edges, i.e. partly concerning mesenchymal cells and partly tissue-specific cells dependent on the involved organ system [2].
Wound healing is an integrated and complex process involving a large number of regulatory molecules, including proinflammatory cytokines and growth factors, and an orchestrated tissue response. Dysregulation in cytokine or growth factor expression dramatically alters the normal wound healing process, and blocking the inappropriate production of specific proinflammatory cytokines or supplementing the milieu with increased quantities of growth factors has demonstrated the central role played by these mediators. Although there has been some success with these approaches in both experimental models and in patients, only through a better understanding of the complexity and diversity of the wound healing process, as well as an improved comprehension of the time-dependent and concentration-dependent responses to individual proinflammatory cytokines or growth factors [3].
Hu et al. [4] investigated whether interleukin (IL)-1 could affect chemokine production by two different types of cells that are present within wounds and found the level of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 1α, exhibited very large (75-and 463-fold, respectively) differences within wound tissue and genetic variation within Nalp1, an inflammasome component that regulates IL-1 production, correlated with such difference, and consistent with the genetic correlation, IL-1β was shown to stimulate KC production by murine keratinocyte and fibroblast cell lines in vitro IL-6 is produced in the wound by epidermal keratinocytes, dermal fibroblasts, and macrophages and it affects multiple processes that are related to wound healing. After wounding, IL-6 is produced and it increases adhesion of neutrophils to dermal fibroblasts and modulates growth factors. IL-6 expression is intimately involved in reepithelialization, granulation tissue formation, and inflammation. Preliminary data suggest that the mechanism by which IL-6 controls wound healing is indirect; involving the regulation of genes involved in growth factor expression a processes that need its presence for long duration [5].
After surgical trauma, burns or injury, a reduction of cellular proliferation and secretion of cytokines by mitogen-stimulated T lymphocytes has been observed, consistent with this, there is defect of stimulated T cells to proliferate and to secrete TNF-α. Interestingly, TNF-α and interferon-γ deficiencies have been shown to result in impaired immune defense against protein antigens and various pathogenic microorganisms [5,6].
The current comparative study aimed to evaluate the ability of estimation of tissue extract fluid levels of IL-1β, TNF-α and IL-6 for differentiation between antemortem and postmortem wounds and as a trial for rough determination of time lapsed since wound inflection.

Patients
The current study was conducted at Departments of Forensic Medicine and Medical Biochemistry, Faculty of Medicine in conjunction with General Surgery Department, Benha University Hospital since Jan 2011 till Sep 2012. After approval of the study protocol by the Local Ethical Committee and obtaining written nearest patients' relative consent, 20 patients arrived to Surgical Emergency Department with severe trauma requiring surgical intervention and died after a known survival interval were enrolled in the study. Patients discharged alive or those died immediately on arrival to ED were not enrolled in the study. Also, diabetic patients and patients maintained on corticosteroids, immunosuppressive drugs or anti-inflammatory drugs were excluded.

2.2: Methods 2.2.1: Specimen collection
Just at time of arrival to the ED, the time elapsed since trauma inflection till arrival to the ED and the presence of multiple trauma were determined. Four skin biopsies were obtained from each patient: 1. Antemortem (AM) specimens were obtained just at time of surgical interference and time was determined since arrival to ED till transfer to theater and its sum with time since trauma inflection equals age of the wound. 2. Postmortem (PM) specimens included sample obtained from wound edge 30 minutes after death (PM1), from the contralateral side of the site of the wound 30 minutes after death (PM2) and from the wound edge 6 hours after death (PM3).
Wound specimens were taken parallel to the wound margin and measured approximately 0.5-1.0 cm in length and 0.2-0.3 cm in width. The subcutis was removed and skin samples were stored at −70°C until analysis.

2.2.2: Specimen processing
1. Tissue extraction: the frozen skin specimens were homogenized mechanically and by incubation in an oscillating mill under the permanent addition of liquid nitrogen (1-2 min). The resulting product, which resembled a powder, was transferred to cups, weighed (wet weight) and extracted in a ten-fold volume of phosphate buffered saline (PBS, pH 7.4) and protease inhibitors [2 mM phenylmethylsulfonyl fluoride (PMSF); 5 mM ethylenediaminetetraacetate (EDTA); 1µg/ml of leupeptin, antipain, aprotinin and pepstatin A, respectively]. The extraction took 1 h at 4°C under permanent agitation. Then the samples were centrifuged for 20 min at 15000 g and 4°C.
The resulting solution (soluble cytokine fraction) was stored at −70°C until ELISA analysis [7]. 2. Determination of sample protein content (mg/ml) by three-fold photometric measurements at 560 nm according to the microtiter plate method of the bicinchoninic acid protein assay (Pierce, USA) [8].

2.3: Statistical analysis
Obtained data were presented as mean±SD, ranges, numbers and percentages. Results were analyzed using Wilcoxon; ranked test for unrelated data (Z-test) and Chi-square test (X 2 test). Possible relationships were investigated using Pearson linear regression. Sensitivity of estimated parameters as predictors for vitality were evaluated using the receiver operating characteristic (ROC) curve analysis judged by the area under the curve (AUC) compared versus the null hypothesis that AUC=0.05. Statistical analysis was conducted using the SPSS (Version 15, 2006, SPSS Inc., Chicago, IL, USA) for Windows statistical package. P value <0.05 was considered statistically significant.

Results
The study included 20 patients, 16  Mean TEF levels of IL-1β and TNF-α estimated in AM specimens were significantly (p<0.05) higher compared to their levels estimated in the three PM specimens. Moreover, mean TEF levels of IL-1β and TNF-α estimated in PM1 and PM3 specimens were significantly (p<0.05) higher compared to their levels in PM2 specimens with non-significant (p>0.05) difference between PM1 and PM3 specimens, (Fig.  1). On contrary, mean TEF levels of IL-6 estimated in PM2 specimens were significantly (p<0.05) higher compared to the levels estimated in AM, PM1 and PM3 specimens. Mean TEF levels of IL-16 estimated in AM specimens were significantly (p<0.05) higher compared to levels estimated in PM3 specimens, but were non-significantly (p>0.05) higher compared to levels estimated in PM1 specimens with non-significant (p>0.05) difference between PM1 and PM3 specimens, (Table 1, Fig. 2).
For differentiation between AM and PM specimens, there was a positive significant correlation between TEF levels of IL-1β and TNF-α with vitality of the wound, while the correlation was negative and nonsignificant between wound vitality and TEF levels of IL-6. On the other hand, TEF levels of IL-6 showed a positive significant correlation with time lapsed since wound inflection, while such correlation was positive but non-significant with TEF levels of IL-1β and TNF-α, ( Table 3).
Using ROC curve analysis, elevated TEF level of IL-1β showed high sensitivity for identification of wound inflected since 60 minutes or less with significant difference of AUC compared versus the null hypothesis, (AUC=0.186, p=0.035) while TNF-α and IL-6 showed non-significant difference (AUC=0.334 & 0.401, p>0.05, respectively) versus the null hypothesis as sensitive predictors of similar wounds (Fig. 3).

Discussion
Skin wound healing is a primitive but well orchestrated biological phenomena consisting of three sequential phases, inflammation, proliferation, and maturation. Wound examination is indispensable in forensic practice; it is always necessary to determine wound vitality or wound age to correctly evaluate the relationship between death and any wounds. With the development of immunohistochemistry and chemical analyses, the scientific field of wound age determination has advanced progressively during recent years. In particular, it has been demonstrated that collagens, cytokines, and growth factors are useful candidates and markers for the determination of wound vitality or age [12,13].
The current study aimed to evaluate the ability of estimation of TEF levels of certain inflammatory cytokines for differentiation between antemortem and postmortem wounds and as a trial for rough determination of time lapsed since wound inflection. In line with the methodology applied regarding estimation of TEF cytokines and not in serum, Carvalho et al. [14,15] found no correlation between wound exudates levels of IL-6, IL-1β and TNF-α and other cytokines and their serum levels in patients undergoing surgical procedure and concluded that the lack of significant correlations between wound and serum levels emphasizes the importance of determining site-specific release.
In line with the target of work, Guler et al. [16] tried to determine the importance of ubiquitin and tenascin in wound age and found that tenascin and ubiquitin together was useful in determining wound age semiquantitatively. Ishida et al. [17] examined the expression of cyclooxygenase-2 (COX-2) using 60 human skin wounds of different ages and reported that collectively, COX-2 would be a useful marker for the determination of early wound age Mean AM specimens TEF levels of IL-1β and TNF-α were significantly higher compared to their levels estimated in the three PM specimens with significantly higher levels in PM1 and PM3 specimens compared to PM2 specimens. Mean PM2 specimens TEF levels of IL-6 were significantly higher compared to AM, PM1 and PM3 specimens with significantly higher levels in AM specimens compared to PM3 specimens.
In line with these data, Grellner et al. [18] found IL-1β, IL-6 and TNF-α were weakly expressed in normal human skin, however, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands and showed enhanced expression after 15 and 20 min at the earliest increase of epidermal reactivity and after 30-60 for IL-1β and 60-90 min for IL-6 and TNF-α, marked expression was observed, persisted over several hours and then decreased to basal levels again and concluded that proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early posttraumatic interval prior to leukocyte reaction. Wang & Ding [19] explored the relationship between the change of cytokines levels and the wound age during the healing process of rats skin wound using ELISA assay performed on intravital skin wounds after incision 0.5-168 h to detect their dynamics expression and reported that the level of IL-2 and TNF-α increased at 30 min after wounding, got to a peak at 3 h for IL-2 and 1 h for TNF-α after injury, rebound of levels were shown at 48 h after wounding, and levels were inclined thereafter.
Takamiya et al. [20] performed quantification of IL-2, 4, 6, 8 and 10, IFN-γ and TNF-α in human dermal wounds for wound age estimation and found that among the cytokines analyzed IL 6, IL 8, IFN-γ, and TNF-α were strongly expressed and suggested that multiplex cytokine analysis at the wound site can be useful for wound age estimation. Thereafter, Takamiya et al. [20] tried wound age estimation for human dermal wounds based on quantification of 9 cytokines and found the set of cytokines selected wound be useful in daily forensic practice.
The significantly higher levels of the estimated cytokines in AM specimens compared to PM3 specimen indicated the fact that the transudation of the tissue with blood components (soluble cytokines in the serum, additional release from cellular elements such as monocytes) may contribute to the rapid quantitative increase of proinflammatory mediators and the negative results in PM wound point to the fact that intact circulation is required and that sole passive transudation occurring in postmortem injury is not sufficient to raise tissue cytokine levels [21]. However, the significantly higher levels of IL-6 in PM2 wound specimens; i.e., wound inflected postmortem could be attributed to the fact that multipotent keratinocytes, mast cells, sweat glands and partly macrophages contain higher amounts of various cytokines especially IL-6 and are stored there as inactive precursors or in the active form thus acting as a continuous source for this cytokine for a longer duration [22], (Holzheimer & Steinmertz, 2000).
There was a positive significant correlation between TEF levels of IL-1β and TNF-α with wound vitality, while TEF levels of IL-6 showed a positive significant correlation with time lapsed since wound inflection. ROC curve analysis defined elevated TEF level of IL-1β as a sensitive indicator of vitality during wound inflection and assured wound age of ≤60 minutes. This finding could be attributed to the fact IL-6 production requires the expression and release of IL-1 so as long as the tissues are viable there is more IL-1 and subsequent increase of IL-6 production. In line with this attribution Sugawara et al., (2001) reported that constitutive keratinocytederived IL-1 is a stimulus for IL-6 production in wounded epidermis and the response involves nuclear factor (NF) kappa B and NF-IL-6 transcription factors.
The obtained results and review of literature allowed concluding that estimation of TEF levels of IL-1β, TNF-α and IL-6 could differentiate between wounds inflected while victim was alive and PM wound. However, estimation TEF level of IL-1β works better for such differentiation and could determine wound inflected within 60 minutes with high sensitivity.   .001 Data are presented as mean±SD, ranges are in parenthesis p1: significance of difference between TEF levels in AM and PM1 specimens p2: significance of difference between TEF levels in AM and PM2 specimens p3: significance of difference between TEF levels in AM and PM3 specimens p4: significance of difference between TEF levels in PM1 and PM2 specimens p5: significance of difference between TEF levels in PM1 and PM3 specimens p6: significance of difference between TEF levels in PM2 and PM3 specimens