HISTOLOGICAL STUDY AND IMMUNOHISTOCHEMICAL EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR IN SKIN WOUND HEALING AND ITS APPLICATION FOR FORENSIC WOUND AGE DETERMINATION

Wound healing examination is one of the most important aspects of the forensic pathology to estimate wound age. Chronological histopathological alterations characterizing the different phases of wound healing can be applied to wound age determination. Recently immunohistochemical studies are used for cutaneous wound age determination. Therefore, the aim of this work wasto investigate the morphological changes as a result of ante-mortem (A/M) and post mortem (P/M) incisional skin wound and to evaluate the immunohistochemical expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) for wound age estimation. Thirty five adult male albino rats were divided into seven groups (five rats each). group1 (G1) five rats was used as a control to study the normal skin structure. Twenty five rats were wounded; one cm length and full thickness skin incision was made on the back of each animal. The animals were then divided into five equal groups. Skin biopsies were taken 6 hours (G2), 1 day (G3), 3 days (G4), 6 days (G5) and 10 days (G6) after incision. G7 represented P/M group. Skin biopsies were processed for histological and immunohistochemical studies. Morphometric and statistical analysis were done. Different degrees of wound healing signs (re-epithelization, granulation tissue formation, cellular infiltration and new collagen formation) could be detected with L/M examination of H&E and masson trichrome stained sections of A/M wounded groups. In P/M wound, no signs of wound healing could be detected. Immunohistochemical study of all groups revealed that iNOS expression markedly increased after 6 hours and peaked at 1day after incision. After 3days and 6days, it is still highly significant increased but less than 6hours after incision and peaked again after 10days to be nearly of its expression at 6hours after incision. iNOS was not expressed in P/M wound. VEGF expression gradually increased with increase post wounding time, it peaked at 10 days after incision but was not expressed in normal intact skin, 6 hours after incision and in P/M wound. There was highly significant correlation between optical density means of iNOS / VEGF and optical density means of VEGF/mean area % of collagen fibers. We can conclude that, time dependent expression of both iNOS, VEGF proteins and area % of collagen fibers suggested that they would be useful markers for the determination of cutaneous wound age assisted by histological changes during different stages of wound healing.