الفهرس 
Aim of The WorkOnly 14 pages are availabe for public view
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Abstract
The wound is defined as the morphologic functional disruption of the continuity of a tissue structure.
Determining the age of a wound is challenging in forensic medicine, but it can contribute to the reconstruction of crime scenes and lead to arrest of the suspects. Currently, the wound age is a principal parameter in forensic investigations.
Skin wound healing is considered a complicated and well-organized biological response composed of three phases including inflammation, granulation tissue formation, and remodeling, which involve large numbers of regulatory molecules, like cytokines and growth factors.
A variety of methods for estimation of the wound age have been established, such as routine histopathological examination, immunohistochemical staining, and reverse transcription polymerase chain reaction (RT-PCR).
Because wound-age estimation is a complicated and multifactorial problem thus, the use of a combination of several parameters could reduce the errors in wound-age estimation.
The present study aimed to evaluate the role of biomarkers in determination of wound age at different times in rats through the following objectives:
1. To detect histopathological examination of skin wound at different times.
2. To determine the vascular endothelial growth factor (VEGF), alpha-smooth muscle actin (α-SMA) by immunohistochemical examinations and tumor necrosing factor alpha (TNF-α), transforming growth factor (TGF-β1) through gene expressions in rat skin wounds at different ages.
3. To assess the practical availability of the above markers for its forensic application as markers for wound age determination.
This study was conducted on 48 healthy adult male albino rats, weighting from 150 to 200 g. They were obtained from the animal house of the Faculty of Veterinary Medicine, Benha University. Experimental procedures were performed according to the guidelines for the care and uses of laboratory animals approved by Research Ethics Committee (REC), Faculty of Medicine, Benha University.
Rats were selected randomly and divided into 8 equal groups, six rats for each group: Control group I, wound groups (group II, III, IV, V, VI, VII& VIII) in which the rats were sacrificed at different time intervals (12 hours, 1st day, 3rd day, 5th day, 7th day, 10th day& 14th day) after wounding respectively.
The animals were anesthetized and the hair on the dorsum of each rat was shaved leaving a bare region with a diameter of 2 x 2cm then a 2-cm full-thickness incision was made on the bare area in wound groups (group II, III, IV, V, VI, VII& VIII). After injury, rats were sacrificed after being anesthetized at the following intervals respectively 12 h, 1d, 3 d, 5 d, 7 d, 10 d, 14 d (one group at each interval). Also, the skin specimens of six unwounded rats (control group) were also examined.
The wound biopsies were immediately fixed in neutral buffered formalin (10%) for 24-48h routinely processed and paraffin embedded sections on glass slides were prepared for staining with Hematoxylin and Eosin (H&E) for histopathological examination and immunohistochemical staining for (VEGF and α-SMA) while other skin sections kept in glass Teflon for RNA extraction and RT-PCR gene expression of (TNF-α and TGF-β1).
Data were statistically analyzed and revealed the following results:
The results of the histopathology study revealed that:
The histopathological examination of skin tissue sections at 12 hours after wounding (group II) showed ulceration of the epidermis and disturbance of dermis structure and disarrangement of the fibers, lost areas are also seen.
At 1 day after inducing the wound (group III), histopathological examination of skin tissue sections showed degeneration of dermis and inflammatory cellular infiltration, degeneration of the glands and blood vessel congestion are also seen.
At 3 day after inducing the wound (group IV), histopathological examination of skin tissue sections showed reepithelialision of the epidermis, fibroblast appear with flat nucleus , monocellular infiltration and granulation tissue formation.
At 5 day after inducing the wound (group V), histopathological examination of skin tissue sections showed reepithelialision of the epidermis, fibroblast appear with flat nucleus, sever monocellular infiltration and granulation tissue formation and neovascularization. The dermis showed a cut section of the shaft of the hair follicle.
At 7 day after inducing the wound (group VI), histopathological examination of skin tissue sections showed good reepithelialision of the epidermis, neovascularization, the dermis showed proliferation of the fibroblasts and well organized fibers and fibroblast appear with flat nucleus for collagen fibers deposition, sever monocellular infiltration and granulation tissue formation.
At 10 day after inducing the wound (group VII), histopathological examination of skin tissue sections showed good reepithelialision of the epidermis, neovascularization, sever monocellular infiltration and granulation tissue formation and proliferation of fibroblast. Fibroblast appears with flat nucleus for collagen fibers deposition and well organized fibers.
At 14 day after inducing the wound (group VIII), histopathological examination of skin tissue sections showed the epidermis covered with a layer of keratin, new hair follicle and multiple glands.
- The results of the immunohistochemical study revealed that:
• Immunohistochemical examination of all skin sections in control group (group I), group II (skin samples were taken at 12h after inducing the wound) and group III (skin samples were taken 1 day after inducing the wound) showed mild α-SMA and VEGF expression.
• Whereas, immunohistochemical examination of all skin sections in group IV (skin samples were taken 3 day after inducing the wound), group V (skin samples were taken 5 day after inducing the wound) and group VI (skin samples were taken 7 day after inducing the wound) showed moderate α-SMA and VEGF expression.
• As well, immunohistochemical examination of all skin sections in group VII (skin samples were taken 10 day after inducing the wound) and group VIII (skin samples were taken 14 day after inducing the wound) showed severe α-SMA and VEGF expression.
• Comparison between different groups regarding intensity of vascular endothelial growth factor (VEGF) and α-smooth muscle actin (α-SMA) immunohistochemical expression showed that there were highly significant differences between the means of each of VEGF and α-SMA at the different time points in the studied groups.
• There was positive correlation (significant increase) between VEGF and SMA morphometry and time of wound. Also, simple linear regression showed that VEGF and α-SMA markers are highly significant predictors of wound time (p <.001).
The results of mRNA expression of tumor necrosing factor alpha (TNF-α) and transforming growth factor (TGF-β1) genes revealed that:
• There was a highly significant increase in expression of both TNF-α and TGF-β1 in group II, III, IV, V and reached to the peak in group VI then a sharp downregulation was detected in the group VII, VIII.
• Comparison between different groups regarding relative quantity (R Q) of mRNA expression of TNF-α and TGF-β1 markers showed that there were highly significant differences between the means of each of TNF-α and TGF-β1 at the different time points in the studied groups.