Time-dependent Changes Of INOS And ENOS Protein Expression In Rat Cutaneous Incised Wound Healing

Objective: The aim of this study was to observe the changes of inducible nitric oxide synthases (iNOS) and endothelial nitric oxide synthases (eNOS) protein expression during rat cutaneous incised wound healing, attempting to find out the feasibility applying to wound time determination in forensic practice. Methods: After setting up the model of rat cutaneous incised wound, iNOS and eNOS protein were observed with immunohistochemical technique and the method of imaging analysis during rat cutaneous incised wound healing (1h to 14day after wound). Effect of postmortem intervals from 6h to 48h on iNOS and eNOS protein expression was also studied. Results: (1) Changes of iNOS protein expression during wound healing: Immunohistochemically, iNOS protein was negative in normal rat skin. After incised wound, iNOS protein was stained brown-yellow in the cytoplasm. iNOS protein was not only detected in the cytoplasm of keratinocytes, fibroblasts, endothelial cells and inflammatory cells but also in sweat glands, hair follicles and skeletal muscles in injuried skin. Changes of iNOS protein expression were studied by cutaneous and subcutaneous histological part respectively.â‘ Changes of iNOS protein expression in granulation tissue during wound healing: Low positive staining was observed in macrophages and neutrophils at initial 6h after wound. The number of positive macrophages and neutrophils was increased immediately at 12h. Marked iNOS protein expression was showed at 1 day and sustained at a high level up to 5 day after wound, which was observed in inflammatory cells, fibroblasts and endothelial cells in granulation tissues. Thereafter, the number of positve cells declined and the degree of staining was also weakened from 7 day to 14 day after wound. â‘¡Changes of iNOS protein expression in epidermis during wound healing: Keratinocytes expressed low-level iNOS at initial 1h after wound. Strong positive staining was observed at 12h and peaked at 3 day after wound, with high expression up to 7 day after wound. Thereafter, the degree of staining steadily declined from 7 to 14 day after wound and keratinocytes expressed low-level iNOS protein. â‘¢Changes of iNOS protein expression in sweat glands during wound healing: Sweat glands expressed low-level iNOS protein at initial 1h, too. With the extension of wound time, the expression of iNOS protein in sweat glands gradually increased and reached to a high level from 1 to 5 day after wound. Strong positive staining was detectable in sweat glands. Subsequently, there was a steady decline from 7 to 14 day after wound. â‘£Changes of iNOS protein expression in skeletal muscles during wound healing: Low positive staining in skeletal muscles was detected at initial 1h after wound. With the extension of wound time, the expression of iNOS protein in skeletal muscles wasslowly increased and peaked at 7 day after wound. Subsequently, there was a slow decline up to 14 day after wound. Consequently, the changes of iNOS protein expression in epidermis, sweat gland, granulation tissue and skeletal muscle were time-dependent during wound healing. There was most marked trend in granulation tissue compared with each other, which reflected the relationship with wound time preferablely. Secondly, it was in sweat glands. The expression of iNOS protein in epidermis was highest, but the variational trend was small. Skeletal muscles only expressed comparatively low iNOS protein during wound healing, which made it small significance for estimating wound time. (2) Effect of postmortem intervals on iNOS protein: Effect of postmortem intervals from 6h to 48h on iNOS was observed in 1d's, 3d's and 5d's injury. The degree of positive staining was decreased with the extention of postmortem intervals. There were no remarkable changes in degree of iNOS protein staining of epidermis, sweat glands and skeletal muscles from postmortem 6h to 12h, but declined remarkablely from postmortem 24h to 48h compared with 3d's injury. At postmortem 6h, there were no remarkable changes in granulation tissue for iNOS pro...