| Abstract |
Two zones may be histologically distinguished around the antemortem wound. A central zone where a decrease in the vitality of the cells of the connective tissue (negative vital reaction) appears in the immediate vicinity of the wound edge (Raekallio 1972, 1980, 1984). This comes as a consequence of the mechanical damage caused by the injury, and the reduction of the blood supply caused by the local destruction of the blood vessels and inflammation. The cells of that area show a progressive loss of enzyme activity 1-4 hours following the injury infliction. All the above are considered as early signs of the forthcoming necrosis in the central zone of the trauma. In the peripheral zone, a significant increase of the enzyme activity is exhibited (positive vital reaction). Both the activity and the quantity of the enzymes increase as shown by quantitative measurements of aminopeptidases and phosphatases (Raekallio 1972, Gallo 1997). This increase comes from the cells in the peripheral zone of the traumatic area, from the plasma outside blood vessels and from the lymphocytes that evade the area. In total all the above are part of the defense system of the body in cases of injury (Raekallio 1972, 1980, Gallo 1997). The determination of wound age as well as the distinction between antemortem and postmortem wounds, both in human and animals, is one of the most important medico legal problems, up to now (Benz 1994, Raekallio 1972). During wound healing, serious histological and enzyme processes take place, which are visible microscopically and histochemically. Researchers that dealt with human wound age estimation (Benz 1994, Raekallio 1972, 1970, 1980, Berg 1972) have used several histochemical, biochemical, histological and immunohistochemical methods. The histological methods determine the age of the wound according to the chronological order of appearance of the cellular elements (neutrophili, granulocytes, macrophages, lymphocytes, etc) (Benz 1994, Raekallio 1972, 1984, Perper et al 1980). The histological methods are simple to apply but have the disadvantage of giving clear results 8-16 hours (h) after the infliction of the injury. The histochemical methods defined the wound age according to the changes of enzyme activity in the wound area. The biochemical methods determine the values of histamine and serotonin and the immunohistochemical methods may also be used to determine the age of the human skin wound (determination of Fibronectin, Tenascin, Collagen type I, III, VI and V) (Dachum et al 1992, Benz 1995, Hausmann et al 1997). Treatment of experimental animals Eighty New Zealand white rabbits, (50 females, 30 males) one to 42 months old were used (figure 1). Incisions 1.0 to 2.0 cm long were inflicted on the skin of rabbits after local anaesthesia. The specimens were taken after euthanasia or under general anaesthesia. A total of 505 specimens of skin wounds were examined, of which 410 were antemortem and 95 were postmortem skin wounds. The post infliction interval ranged from 0.5 h to 144 h (0.5h, 1h, 1.5h, 2h. 2.5h, 3h, 3.5h, 4h, 6h, 12h, 20h, 24h, 32h, 48h, 72h, 96h, 120h, 144h), in the antemortem skin wounds. The postmortem skin wounds were inflicted from 0.5h to 4h (0.5h, 1h, 2h, 3h, 4h) after death. The specimens from the 95 postmortem skin wounds were removed at time intervals ranging from 1h to 32h after postmortem infliction (1h, 2h, 3h, 3.5h, 12h, 20h, 24h, 32h). The specimens were divided into 3 groups. The first group contained 360 antemortem skin wounds in which the specimens were removed immediately after the death (or anesthesia) of the experimental animals. The second group contained 50 antemortem skin wounds in which the specimens were removed from 6h to 72h after the death (or anesthesia) of the experimental animals. The third group contained specimens from 95 postmortem skin wounds The specimens were frozen in liquid nitrogen immediately after taking. The specimens were cut in sections of 10 microns in cryostat at -20oC and right after without fixation the histochemical examination was performed (Fujimoto et al 1997, Sony et al 1996). The enzymatic activity of nonspecific esterases, adenosine triphosphatase (ATPase) and alkaline phosphatase was investigated. Additionally each specimen was stained by hematoxylin-eosin to detect the typical morphology of the lesions in the wound area. The control specimens comprised of tissues from injuries incubated without substrate. Alkaline phosphatase A total of 300 antemortem skin wounds were examined. The azo dye coupling method was used for the histochemical determination of alkaline phosphatase activity (Bancroft 1977, Espada et al, 1998). Reagents: sodium a-naphthyl phosphate 10 mg, Tris buffer 0.1M (stock solution), pH 10.0 10 ml, diazonium salt fast red TR 10 mg. The pH of the incubating medium was 9.2. The specimens were incubated at room temperature for 10-60 min, were washed in distilled water, were counterstained in 2 % methyl green solution (chloroform extracted), washed with running tap water and mounted in glycerin jelly. Alkaline phosphatase activity appeared as a reddish-brown stain. Nonspecific esterases A total of 250 antemortem skin wounds were examined. The Nachlas and Seligman method modified by Pearse (Raekallio 1972, 1980) was used for the histochemical determination of nonspecific esterases activity. Reagents: sodium a-naphthyl acetate 10 mg, acetone 0.25 ml, phosphate buffer 0.1 M (stock solution), pH 7.4 10 ml and diazonium salt fast blue B salt 50 mg. The specimens were incubated at room temperature for about 5 min, washed in distilled water for 5 min and mounted in glycerin jelly. The nonspecific esterases activity appeared as a reddish-brown stain. Adenosine triphosphatase (ATPase) A total number of 200 antemortem skin wounds were examined. The Wachstein and Meisel method modified by Pearse (Bancroft et al 1977) was used for the histochemical determination of ATPase activity. Reagents: ATP disodium salt 2 mg, Tris buffer 0.2 M, pH 7.2 0.4 ml, lead nitrate 2 % 0.1ml, magnesium sulphate 2 % 0.1 ml, distilled water 0.5 ml and 2,4-dinitrophenol 1.5 mg. The specimens were incubated at 37oC, for about 60 min, washed in distilled water, immersed in 1 % ammonium sulfide for 1 min, rinsed under running tap water and were mounted in glycerin jelly. ATPase activity appeared as a brownish-black deposit. The enzyme activity of alkaline phosphatase, of nonspecific esterases and ATPase was determined in the postmortem skin wounds according the histochemical methods used for the determination of antemortem skin wounds. Antemortem wounds: A number of 410 antemortem wounds were examined. Alkaline phosphatase: In normal skin the epidermal layer showed no staining. In the skin appendages, vessel walls and dermal fibroblasts, positive reactions were remarked. In the vital skin wounds, the increase of alkaline phosphatase activity appeared about 3.5 h after wounding. Maximum enzyme activity was reached for alkaline phosphatase about 32 h after wounding while 2 % of the examined specimens gave a negative result (table 1, figure 2). Nonspecific esterases: In normal skin there was an intense site of esterases activity between the stratum granulosum and stratum corneum. The root sheaths, the upper bulbs of the active hair follicles and the dermal fibroblasts showed strong reactions. In the vital skin wounds, the increase of nonspecific esterases activity appeared about 1 h after wounding. Maximum enzyme activity was reached for nonspecific esterases about 24 h after wounding while 1.2 % of the examined specimens gave a negative result (table 1, figure 2). ATPase: In the normal skin, the skin appendages, vessel walls, musculi arrectores pilorum and dermal fibroblasts, gave positive reactions. A moderate reaction was detectable in the stratum granulosum and stratum basale. In the vital skin wounds, the increase of ATPase activity, appeared about 2 h after wounding. Maximum enzyme activity was reached for ATPase about 20 h after wounding while 1.5 % of the examined specimens gave a negative result (table 1, figure 2). Fifty sections of the 410 total antemortem skin wounds were sampled for the enzyme histochemical examination 6 h to 72 h after death (or anesthesia), which also exhibited changes of the activity of alkaline phosphatase, nonspecific esterases and ATPase similar to the 360 antemortem skin wounds.
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