Antemortem And Postmortem Estimation Of Rat Wounds Biology Essay

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Introduction: Inflammatory mediators play an important role in the mediation of inflammation and trauma. There for they could be useful for the determination of vitality and wound age.

Objective: In the present study, interleukin-6 (IL-6), fibronectin (Fn) and lipoxinA4 (LXA4) were estimated in extracts of antemortem skin wounds in rats. Moreover, we extended our measurements to include the levels of these mediators in rat skin wounds in the early postmortem period aiming to test for their practical usefulness in the estimation of wound vitality and the duration after its infliction.

Methods: Thirty two rats were divided into 4 groups of rats (8 animals in each group) and were assigned for collection of wound samples at the indicated time intervals. The wound samples were taken 30 min (group I), 3 hours (group II), 6 hours (group III) and 24 (group IV) hours after infliction of the incised wounds. The specimens as control group were excised from uninjured rats (8 animals) in the same region as wounded groups. The rats in group IV were sacrificed by cervical dislocation and kept at room temperature to be used in assessment of postmortem changes on different parameters. Postmortem wound samples were then collected 24 hours after sacrifice of group IV rats and the postmortem control samples were taken from intact skin of the same rats. A special group of rats (n=8) was used to explore the influence of supravital injuries on mediator release from postmortem inflicted wounds. All wound and control specimens were homogenated and assayed for the level of IL-6, Fn and LXA4 using quantitative ELISA analysis.

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Results: Analysis of the changes of the studied parameters in different times revealed that Fn is the first mediator to increase, in 30 min, after wound infliction. In the following 3 and 6 hours after wounding both Fn and IL-6 were increased. At 24 hours of wounding LXA4 increases to join Fn and IL-6, at that time Fn and IL-6 were still high. This pattern of increased level of the three mediators was maintained for at least 24 hours postmortem.

Conclusion: We can conclude that the combined assay of IL-6, Fn and LXA4 may be a useful tool in determination of the probable duration lapsed since antemortem wound inflection. Moreover, this pattern of time dependent increase of the three parameters may be also useful in age determination of multiple inflicted wounds at variable intervals in the same victim. We can also conclude that vital reactions are essential for release of the assayed parameters. This can be documented by the lack of significant increase of these parameters in postmortem-

inflicted wounds.

Keywords: Antemortem, postmortem, wound age.

INTRODUCTION

In forensic practice, it is needless to say that wound examination is one of the most important and indispensable areas for forensic pathologists. During wound examination, it is always required to discriminate antemortem wounds from postmortem damage.(1)

Postmortem injuries can occur accidentally

during handling of a corpse, in necropsy, or caused by animals. Intentional postmortem injuries would be inflicted when trying to hide or suggest a crime, to simulate an accident or a suicide, or to occult

the identity of a corpse.(2) Therefore, diagnosis of

the vitality of the antemortem wounds is one of

the central issues in daily forensic practice. Vitality of wounds is essential to prove that an injury of

the body was caused during lifetime and not postmortem.(3)

ISSN 1110-0834After confirming vitality of the wound, it is necessary to determine the duration it was sustained before death. It is well known that the degree of difficulty of wound age determination increases

with the shortness of the post-traumatic period. Concerning very short intervals, the wound age question is nearly identical with the issue of vitality. The most difficult question is to differentiate between wounds inflicted shortly before and shortly after death. This coincides with processes in the late agony and the early supravital period. The difficulty of estimating wound age in these circumstances is mainly due to the minimal morphological changes that may occur and due to supravital reactions.(2,3)

In forensic medicine, wound healing sequences

are considered as a tool to determine wound

vitality and wound age. Skin wound healing starts immediately after injury and consists of three phases: inflammation, proliferation and maturation. During the inflammatory phase, platelet aggregation at the injury site is followed by the infiltration

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of leukocytes such as neutrophils, macrophages,

and T-lymphocytes into the wound site. In the proliferative phase, reepithelialization and newly formed granulation tissue begin to cover the

wound area to complete tissue repair. Angiogenesis is indispensable for sustaining granulation tissue. Many cytokines, glycoproteins, growth factors, proteases, and eicosanoids are closely involved in the wound healing process to complete normal tissue repair after damage.(4,5) Therefore, monitoring of the markers of wound healing can be adopted to determine wound vitality and wound age in forensic medicine.(6)

Cytokines are glycoproteins produced by

many inflammatory cells such as neutrophils, lymphocytes, macrophages, etc. They have

multi-biological functions related to immune, hematopoietic, endocrine, nervous and inflammatory systems. At present, it has been well-known that cytokines, especially IL-6, have close relation with wound healing process.(7,8)

Fibronectin is a glycoprotein component of basement membranes and interstitial connective tissues. It is synthesized by various cell types including fibroblasts endothelial cells, hepatocytes, macrophages and astroglial cells.(9) It plays an important role in cell adhesion and cell migration during wound healing, and promotes phagocytosis by macrophages and fibroblasts.(10) Fibronectin is involved in angiogenesis during wound healing as well.(11)

Eicosanoids are metabolites derived from arachidonic acid (AA). They are inflammatory mediators with chemotactic activity in wound healing. In later phases of wound healing, keratinocytes proliferate and differentiate to cover the exposed wound surface. Fibroblasts and capillaries produce new granulation tissue. These cells are regulated by many bioactive substances including eicosanoids.(1)

Lipoxin (LX) is one of the eicosanoids which is produced by lipoxygenases from arachidonic

acid. It is a trihydroxy fatty acid containing a conjugated tetraene produced by the metabolism

of 15-HETE (15-Hydroxyeicosatetraenoic Acid)

or 15-HPETE15-(hydroperoxyeicosatetraenoic acid) within human leukocytes.(12) LXA4 is involved in several biological functions including leukocyte activation, chemotatic effects, natural killer

cell inhibition, and monocyte migration and adhesion.(13-15) Evidence from animal studies on acute inflammation, dermal wounding, angiogenesis and host defense, as well as human clinical data

and in vitro studies, all indicate that lipoxin is prominently involved in endogenous protective and anti-inflammatory eicosanoid circuits.(16)

Although macroscopic and histological examinations are routinely performed in forensic autopsy, wound examination should be improved and renovated with the advance of medical science. Thus, it is desirable that new diagnostic techniques are applied in forensic practices.(17)

The main problem of postmortem human studies, autopsy samples, is associated with the inexactly known survival time. Animal experiments have the advantage of controlled conditions which makes possible to evaluate different parameters under standardized circumstances. Moreover, sequential biopsies can be taken.

In the present study interlukin-6, fibronectin and lipoxin A4 were estimated in extracts of antemortem skin wounds at different time intervals in rats. Moreover, we extended our measurements to include the levels of these mediators in rat skin wounds in the early postmortem period aiming to test for their practical usefulness in the estimation of wound vitality and the duration after its infliction.

Materials:

A total of 48 albino rats weighing 150-200 gm were included in the study. Animals were kept

under normal conditions of feedings, light and temperature. Food and water were allowed ad libitum. Incisions of the skin were made in animals and wound biopsies were collected under anesthesia. The design of the research was permitted by the ethical committee of the Faculty of Medicine, Alexandria University.

Wound incisions:

Rats were anesthetized using diethyl ether inhalation for 90-120 seconds. After shaving the skin on the dorsal region, the skin was cleaned with sterile saline. Nearly 5 cm full-thickness incision was made on the dorsal skin using sterile scalpels. Complete homeostasis with moist sterile gauze was done and any clots present were gently removed before dressing. The incised wounds were then covered with sterile gauze. The animals were then isolated in separate clean cages. The antemortem wound and control skin biopsies were taken under diethyl ether anesthesia in all the tested groups.

The animals were divided as follows:

Control group (n=8):

The control specimens were excised from uninjured rats in the same region as wounded groups after shaving the dorsal skin.

Antemortem inflicted wound group (n=32):

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Thirty-two rats were divided into 4 groups

(8 animals in each group) and were assigned for collection of wound samples at the indicated time interval in each group. The wound samples were taken 30 min, 3 hours, 6 hours or 24 hours after infliction of the wounds in group I, II, III and IV, respectively.

Group I (n=8): biopsies were collected 30 minutes after wound infliction.

Group II (n=8): biopsies were collected 3 hours after wound infliction.

Group III (n=8): biopsies were collected 6 hours after wound infliction.

Group IV (n=8): biopsies were collected 24 hours after wound infliction.

The rats in group IV were sacrificed by cervical dislocation and kept at room temperature to be used in assessment of postmortem changes on different parameters. Postmortem wound samples were then collected 24 hours after sacrifice of group IV rats. Postmortem control samples were taken from intact skin of the same rats.

Postmortem inflicted wound group (n=8):

A special group of rats (n=8) was used to explore the influence of supravital injuries on mediator release from postmortem wounds. The rats in this group were sacrificed and postmortem wounds were inflicted within 10 min after death. The skin samples were then collected 3 hours after the incision.

Wound specimens were biopsied from all animal groups parallel to the wound margins.

Biochemical studies:

All wound and control specimens were homogenated and assayed for the level of interleukine-6 (IL-6), fibronectin (Fn) and lipoxin A4 (LXA4) using quantitative analysis.

Quantitative ELISA analysis (kit and protocol of Assay designs, USA), for IL-6

Quantitative ELISA analysis (kit and protocol of AssayPro, USA), for Fn

Quantitative ELISA technique (Oxford Biomedical Research corporation), for LXA4

Histological studies:

Antemortem and postmortem samples (group IV) were preserved in 10% buffered formol solution (pH 7.4) and sectioned for histologically examination using H&E stains.

METHODS

Tissue extraction for IL6: (18)

The complete extraction was performed

under cooling condition (on ice) because of

the susceptibility of cytokines. The frozen

skin specimens were mechanically homogenized

(1-2 min). The resulting product was weighed

and extracted in a ten fold volume of phosphate buffered saline (PBS,pH 7.4) and protease inhibitors, 5mM ethylenediaminetetraacetate (EDTA); 1ug/ml aprotinin and 1ug /ml pepstatin A. The extraction took one hour at 4°C under permanent agitation. The samples were centrifuged in cooling centrifuge for 20 minutes and the supernatants were collected. The protein concentrations in the resulting solutions were estimated then solutions were stored at -70° C until ELISA analysis.

Tissue extraction for fibronectin:(19)

Fibronectin was extracted from skin specimens with 50mM phosphate buffered saline (pH7.4) containing 1% Triton X-100 and centrifuged at 14000x g for 20 min. The supernatant was collected and protein concentration was measured. The supernatant was stored at -20°C.

Tissue preparation and extraction of lipoxin A4:(20)

Skin specimens were homogenized in methanol (5ml/gm) for 2 min and centrifuged. One ml of the obtained supernatant was diluted with 5ml of H2O and acidified to pH 3.5 with 1N HCl. Columns for extraction of lipoxin A4 (C18 Sep-Pak®light) column (Waters ® Corporation) were preconditioned by washing with 2ml of methanol followed by 2ml of water. Homogenized samples were applied into the preconditioned columns and washed with 5ml of water followed by 5ml of hexane. Lipoxin A4

was eluted from the columns with 2 ml of methyl formate which was then evaporated by a stream

of N2. The residue was dissolved in 200 l of extraction buffer provided by the lipoxin A4 enzyme immunoassay kit (Oxford Biomedical Research Corporation).

Determination of protein content:(21)

Protein content was determined by three fold photometric measurements at 560 nm according to the microtiter plate method of the bicinchoninic acid (BCA) protein assay (Pierce, USA).

Determination of tissue IL-6 by ELISA technique:(22)

IL6 was determined by quantitative ELISA analysis (kit and protocol of Assay disgns, USA) using a microtiter plate reader and an automatic calculation of the results. The result was expressed in pg/mg protein.

Determination of tissue fibronectin by ELISA technique:(22)

Fibronectin was measured by quantitative ELISA analysis (kit and protocol of AssayPro, USA) using a microtiter plate reader and an automatic calculation of the results. The respective fibronectin levels were determined in wound samples and control specimens by means of double measurements .The results had the dimension of pg/mg protein.

Determination of tissue lipoxin A4 by ELISA technique:(22)

Tissue Lipoxin was measured by quantitative ELISA technique (Oxford Biomedical Research Corporation). The respective lipoxin A4 levels were determined in wound samples and control specimens by means of double measurements .The results had the dimension of ng/mg tissue.

Statistical analysis:

Descriptive statistics were calculated as mean

and standard deviation for different mediators. Comparison of the mean mediator values in

the wound samples in different time intervals

was done using analysis of variance followed by Tukey post hoc test for statistically significant differences. While the comparison of antemortem and postmortem samples was done using t and paired t tests. Graphical presentation was done using line graphs. Statistical analysis was done using SPSS version 13.0.

RESULTS

Biochemical Results:

(A) Antemortem inflicted wound group:

Analysis of data of the present study revealed

a significant difference regarding the studied parameter levels at different post traumatic intervals and control samples where (P = 0.0001, 0.0001 and 0.003) for IL-6, fibronectin and lipoxin, respectively (Table I)

Interleukin-6 (IL-6):

Comparison of IL-6 in wounds at 30 minutes following the incision with its level in control skin revealed insignificant difference. Although the level of IL-6 increased significantly after that at 3, 6 and 24 hours (316.30 ± 48.01, 235.13 ± 56.80 and 216.26 ± 49.39, respectively), the significantly highest level was observed at 3 hours (Table I&II and Fig. 1)

Fibronectin (Fn):

Regarding fibronectin, its level increased significantly following incision at 30 minutes

post traumatic (128.27 ± 26.42) in comparison to control samples (60.05 ±13.74), P <0.0001. The level was gradually increasing at 3 hs and 6 hs (153.10 ± 31.27 and 206.19 ± 41.97 respectively) to attain significantly higher levels. The increase in fibronectin level was maintained at 24 hour interval which was insignificant compared to the 6 hour level (Table I&II and Fig.2).

Lipoxin A4 (LXA4):

The level of wound lipoxin did not show a significant increase compared to control level (16.20 ± 9.76) except at 24 hours after the incision (43.61 ± 15.64) (Table I & II, Fig. 3).

Postemortem samples

The levels of IL-6, Fn and LXA4 of control

skin samples at 24 hours postmortem showed insignificant difference in comparison to that of control antemortem skin samples.

Interleukin-6:

IL-6 levels in postmortem wound samples continued to be significantly higher (165.30 ± 43.62) than control samples taken from intact skin

(120.41 ± 29.12). However, the mean difference between wound and control levels of IL-6 at postmortem (44.89 ± 36.37) was significantly lower than that in antemortem samples (101.7 ± 39.21) (Table III and fig. 4).

Fibronectin:

The assay of fibronectin in postmortem wound samples revealed significantly higher levels (181.10 ± 46.58) compared to control levels (57.57 ± 11.01) (Table III and fig.5). The mean difference of fibronectin levels between wound and control postmortem samples (123.57 ± 28.80) showed insignificantly change compared to its mean difference in antemortem samples (148.83 ± 27.95).

LipoxinA4:

Lipoxin level in postmortem wound samples (29.75 ± 10.10) showed significant difference from that of control samples (14.97± 11.79) (Table. III and fig.6). Moreover, mean difference of lipoxin levels between wound and control samples at postmortem (14.78 ± 10.95) showed significant decrease when compared to that at antemortem samples (27.41 ± 12.70).

(B) Postmortem inflicted wound group:

In this group, postmortem wounds were inflicted within 10 min following death, and the level of IL-6, Fn and LXA4 were determined after 3 hours. The comparison of all postmortem levels of three markers with their corresponding control levels showed insignificant increase in their levels (P>0.05) (Table IV).

Histological Results:

Control samples (surgical biopsy):

The intact skin samples showed that the epidermis was very thin and only consisted of epidermal cells with underlying dermis that appear with hair follicles and blood vessels embedded in connective tissue stroma of the dermis. (fig.7)

Wounds at 24 hours (surgical biopsy):

Wounds at 24 hours post infliction of the incision showed hyperplasia and increased thickness of the epithelium at the wound edge. Cellular infiltration into underlying dermis was also noticed. (fig.8)

Control samples (24 h postmortem biopsy):

Twenty-four hours postmortem, little changes of intact skin were seen. Some ballooned cells can be demonstrated. (fig.9)

Wounds at 24 hours (24 h postmortem biopsy)

Twenty- four hours postmortem, the wounded skin showed epithelial changes in the form of loss of cell integrity with loss of normal architecture. Hazy appearance of epithelial cells, increased eosinophelia and shrunken nuclei were also noticed (fig.10)

Table I: IL-6, Fn and LXA4 levels in wounded skin at different time intervals after

antemortem incisions and in control samples (mean±SD)

Time

IL-6 (pg/mg protein)

Mean ± SD

Fn (pg/mg protein)

Mean ± SD

LXA4 (ng/mg tissue)

Mean ± SD

Control

114.56 ± 29.03

60.05 ±13.74

16.20 ± 9.76

30 min

121.76 ± 39.03

128.27 ± 26.42

18.34± 10.54

3 hr

316.30 ± 48.01

153.10 ± 31.27

22.65 ± 12.38

6 hr

235.13 ± 56.80

206.19 ± 41.97

23.97± 13.90

24 hr

216.26 ± 49.39

208.88 ± 42.15

43.61±15.64

ANOVA

P value

17.89

<0.0001*

34.75

<0.0001*

15.03

0.003*

*: Statistically significant at P ≤0.05

Table II: Comparison of IL-6, Fn and LXA4 levels in wounded skin at different

time intervals after antemortem incisions and with control samples.

Group

Compared

to group

IL

(pg/mg protein)

Fn

(pg/mg protein)

LXA4

(ng/mg tissue)

30 min

3 hr

0.0001*

0.53 NS

0.84 NS

6 hr

0.0001*

0.001*

0.99 NS

24 hr

0.003*

0.001*

0.04*

Control

0.45 NS

0.0001*

0.56 NS

3 hr

6 hr

0.01*

0.03*

0.88 NS

24 hr

0.002*

0.02*

0.04*

Control

0.0001*

0.0001*

0.11 NS

6 hr

24 hr

0.65 NS

0.87 NS

0.04*

Control

0.0001*

0.0001*

0.20 NS

24 hr

Control

0.0001*

0.0001*

0.0001*

NS: Not statistically significant

*: Statistically significant at P ≤0.05

Table III: Comparison of IL-6, Fn and LXA4 levels in antemortem and postmortem skin samples

Marker

Time

Wound

(Mean ± SD)

Control

(Mean ± SD)

Difference

(Mean ± SD)

P value

IL

(pg/mg protein)

Antemortem

216.26 ± 49.39

114.56 ± 29.03

101.7 ± 39.21

0.0001*

Postmortem

165.30 ± 43.62

120.41 ± 29.12

44.89 ± 36.37

0.03*

P value

0.05*

0.69 NS

0.009*

Fn

(pg/mg protein)

Antemortem

208.88 ± 42.15

60.05 ± 13.74

148.83 ± 27.95

0.0001*

Postmortem

181.10± 46.58

57.57± 11.01

123.57 ± 28.80

0.0001*

P value

0.24 NS

0.70 NS

0.10 NS

LXA4

(ng/mg tissue)

Antemortem

43.61 ± 15.64

16.20 ± 9.76

27.41 ± 12.70

0.0001*

Postmortem

29.75 ± 10.10

14.97   ± 11.79

14.78 ± 10.95

0.02*

P value

0.05*

0.82 NS

0.003*

NS: Not statistically significant

*: Statistically significant at P ≤0.05

Table IV: Comparison of IL-6, Fn and LXA4 levels in postmortem inflicted

wound with control samples.

Markers

levels

control

(n=8)

Postmortem

inflicted wound

(n=8)

P value

IL-6 (pg/mg protein)

114.56 ± 29.03

125.69 ± 34.10

0.49NS

Fibronectin (pg/mg protein)

60.05 ±13.74

71.50±17.56

0.17NS

LipoxinA4 (ng/mg tissue)

16.20 ± 9.76

22.44±12.40

0.28NS

NS: Not statistically significant

Fig.1: The time-dependent levels of interlukine-6 (pg/mg protein) after antemortem

incisions at different intervals compared to control samples.

Fig.2: The time-dependent levels of fibronectin (pg/mg protein) after antemortem

incisions at different intervals compared to control samples.

Fig.3: The time-dependent levels of LXA4 (ng/mg tissue) after antemortem incisions

at different intervals compared to control samples.

Fig. 4: The mean levels of wound IL-6 after incision in antemortem

and postmortem samples compared to control samples

Fig. 5: The mean levels of wound fibronectin after incision in antemortem

and postmortem samples compared to control samples

Fig. 6: The mean levels of wound LXA4 after incision in antemortem

and postmortem samples compared to control samples

Fig 7: A photomicrograph of intact skin, antemortem, showing thin epidermis with hair follicle (H&E stain, Mic. Mag x400)

Fig 8: A photomicrograph skin at 24 hours after wound infliction, antemortem, showing hyperplastic thickened epithelium at the wound edge with mass of cellular infiltration (H&E stain, Mic. Mag x400)

Fig 9: A photomicrograph of intact skin, 24 hours postmortem, showing more or less normal cells with appearance of some ballooned cells. (H&E stain, Mic. Mag x400)

Fig 10: A photomicrograph of wounded skin, 24 hours postmortem, showing thickened epithelium with loss of normal architecture of cells and hazy appearance. Mass of cellular infiltration is also noticed. Eosinophelia and shrunken nuclei are observed as well (H&E stain, Mic. Mag x400)

DISCUSSION

After traumatic events, different inflammatory mediators are primarily synthesized and / or released at the site of tissue destruction. Systemic levels follow in the later course of the inflammatory

or traumatic process. Therefore, it made sense to quantitate mediator's response to trauma directly in the skin wounds. Local quantitative analysis has

an advantage as many diseases, mainly systemic, may influence the levels of inflammatory mediators in the blood. Therefore, local determination of the mediators in the skin wound prevents theoretically possible falsifications.(23)

For sure it would be ideal to conduct this research on healthy human subjects. However, because of

the ethical restrictions it is difficult to do this. Therefore, the best available alternatives are to conduct experiments concerning wound age estimate on animal skin or human skin during surgery. Many researches done before were performed on human skin excised during surgical treatment of different diseases. The problem with these researches is that the expression of cytokines and other inflammatory mediators would be abnormal because of the influence of the diseases and the changes of internal environment of the tissues. For that reason the obtained results would be unreliable for practical application.(6,24)

In the present study interlukin-6, fibronectin and lipoxin A4 were assayed in the skin of rats as indicators of wound vitality and the time lapsed from the infliction of wounds. The general review of the results of our study showed levels of previous mediators increased in a time - dependent manner after the wounds were inflicted. Moreover, significant differences were observed in their levels at different time points of assessment for each parameter, rendering these mediators suitable for determination of the time lapsed from wound infliction.

Interleukin-6:

In the present study, IL-6 did not show significant increase of its levels except at 3 hours after wound infliction. It attained the highest level at this time but the increase in level was sustained although it was at lower levels in 6 and 24 hours post wound infliction.

Harvima et al(25) study explained the release of

IL-6 firstly by local release from preformed stores. Keratinocytes, mast cells, sweat glands and partly macrophages which are the sources of numerous mediators that are stored in as inactive precursors or in the active form and are ready for release.

Later, the induction of acute phase response to inflammation resulted in IL-6 production by a variety of cells such as leukocytes, monocytes, macrophages, lymphocytes and endothelial cells within few hours in response to inflammatory tissue injury. In addition, the transudation with blood components especially soluble cytokines may contribute to the quantitative increase of the mediators as well. It seems to be evident that all these factors lead to the production of IL-6.(26)

In agreement with our results, Grellner(27) demonstrated an increase of IL-6 after one hour of wound infliction. He also noticed the persistence of this increase up to 5 h and its reappearance in few days after. This difference in pattern of increase of wound IL-6 in comparison to our results could be referred to the different method used for assay. Grellner(27) have used immunohistochemical method for assaying IL-6 that detects mainly cytokines bound on the cell surfaces.

In contrast to the present results, Kondo and Ohshima(28) have observed that IL-6 increased

much later than in our study, although statistical significances were not mentioned. They observed that the maximum levels for IL-6 was not before 12h after wounding.

To simulate the forensic reality, the rats of group IV were left in room temperature for 24 hours. Then, the samples of the same wounds were taken to study the effect of postmortem changes on the different examined markers. We have chosen this group as the three examined parameters showed a significant increase comparable to the other groups.

The assay of IL-6 in the present study 24 hours after animal death showed decrease in its level

than that in antemortem samples although

still significantly higher than control levels.

No significant changes occurred in intact skin. Therefore, this increase can be considered as a vital reaction.

The significant decrease of mean difference of

IL-6 between wound and control postmortem samples than that of antemortem samples may

be explained by the effect of autolysis. The inflammation elicited in wound tissue could be

a cause of acceleration in postmortem changes compared to intact tissues. The accelerated

autolysis was shown by the histological results

as microscopic changes demonstrated loss of

cell integrity and loss of normal architecture. Furthermore, the eosinophelia is attributed to denaturation of cytoplasmic protein with exposure

of basic groups that binds to eosin. The breakdown of cell function results in membrane integrity

loss with release of digestive enzymes.(29) Despite this effect, IL-6 was still significantly higher

than that of control level. It may be explained as stated by Grellner(27) as essential autolysis influence on cytokines could not be markedly observed in absence of putrefaction-changes.

Postmortem inflicted wounds IL-6 showed insignificant change 3 hours after wounding compared to control skin samples. However, some increase was noticed which can be explained by passive transudation of blood components from injured vessels as Grellner et al(30) explained. Therefore, it is apparent that intact circulation is required and that sole passive transudation that may occur postmortem is not sufficient to elevate the level as the vital wound.

Fibronectin:

In the present study, fibronectin (Fn) was the earliest marker that appeared. It increased significantly within the first 30 minutes of wounding. The increase in Fn level was progressive to a maximum at 6 hours. That level was maintained at 24 hour after wound infliction.

The early appearance of Fn in wounds may be due to the rapid accumulation of this extra-cellular matrix glycoprotein in the dermis soon after the injury. This helps in migration of leukocytes in the inflammatory phase of skin wound healing. It also helps in interaction between leukocytes and endothelial cells which is the most important event in inflammation. Furthermore, it is probably derived from damaged blood vessels.(31) All these criteria may explain the early detection of fibronectin rendering it a good marker for wound vitality and wound age determination.

The present results are supported by other studies that reported that recently inflicted wounds showed an immunopositive reaction for fibronectin.(31,32). In agreement with our results, Fieguth et al (33) also demonstrated a strong fibronectin positive reaction in wounds within 30 minutes. Massive reaction was detected in samples older than 3.5 hours. They also demonstrated that a strong positive reaction could still be detected in wounds examined for days.

The present data showed also that the increase of fibronectin was maintained in samples collected 24 hours postmortem. The level of Fn was not significantly different from its antemortem level. This could be explained by the finding that fibronectins have peculiar of anti-autolysis characteristic.(34) For that reason assay of Fn may solve some problems in forensic wound age determination.

However, the sensitivity as well as the specificity of this marker has been questioned. Grellner et al (30) suggested that a fibronectin-positive reaction found in postmortem-incised wounds of porcine skin was similar to those occurring in vital wounds. This finding is contradictory to our results where no significant increase was observed in postmortem inflicted wounds compared to control samples.

However Ortiz-Rey et al (35) supported our results by their observation that a reticular staining for fibronectin appeared in vital specimens but not in postmortem cases. Fieguth et al (33) also did not detect positive reactions in immediately fatal human skin wounds, although extensive lacerations in their examined samples after massive injuries may lead to a more intensive wound reaction than an incision under sterile conditions in our study.

Lipoxin A4:

In the current study, lipoxin showed an insignificant increase except lately 24 hours

after wound infliction. He and Zhu(36) examined

the levels of other related eicosanoids namely leukotrien B4 (LTB4) in human skin wounds

and demonstrated that detecting LTB4 is useful

to distinguish antemortem from postmortem

injuries. However, Hernandez-Cueto et al(37) showed that Prostaglandin F2a (PGF2a) is not suitable to diagnose the vitality of wounds because of its irregular behavior.

Lipoxin level showed significant difference when compared with intact skin 24 hours after death. However it was significantly lower than its level 24 hours after antemorten-inflection of wounds.

This could be explained in the light of the observations of Hart et al(38) who found a decrease in postmortem compared to antemortem lipid levels. They explained this difference by postmortem degradation of lipids. Autolysis may be also accelerated in the wounded skin as a result of inflamation resulting in more degradation in wounded skin rather than the normal tissue.

As the case with other examined mediators in the current study, lipoxin showed insignificant change three hours after postmortem inflicted wound compared to control skin.

Conclusion:

Analysis of the changes of the studied parameters in different times revealed that Fn is the first mediator to increase, in 30 min, after wound infliction. In the following 3 and 6 hours after wounding both Fn and IL-6 were increased. At 24 hours of wounding LXA4 increases to join Fn and IL-6, at that time Fn and IL-6 were still high. This pattern of increased level of the three mediators was maintained for at least 24 hours postmortem. Taking in consideration the aforementioned data we can conclude that the combined assay of IL-6, Fn and LXA4 may be a useful tool in determination of the probable duration lapsed since antemortem wound inflection. Moreover, this pattern of time dependent increase of the three parameters may be also useful in age determination of multiple inflicted wounds at variable intervals in same victim.

We can also conclude that vital reactions

are essential for release of the assayed parameters. This can be showed by the lack of significant increase of these parameters in postmortem-inflicted wounds.