Epidermis of amputated limbs in patients with diabetes mellitus - immunohistochemical study

Aim. To examine the skin morphological changes in different areas of the amputated lower limbs of patients with diabetes mellitus. Methods. Skin samples of lower limbs of 6 patients with diabetic foot syndrome were studied using immunohistochemistry methods. Proliferation of epidermal cells, their resistance to apoptosis, as well as the degree of cells differentiation (using antibodies to proliferating сell nuclear antigen, Bcl-2 anti-apoptotic activity marker, epidermal cells terminal differentiation marker cytokeratin 1) were assessed. The color intensity was assessed using ImageJ software and was displayed as «mean greyscale intensity» (an integral value summarizing color intensity of three basic colors - red, green and blue - by V=0,299R+0,587G+0,114B formula, where V - mean intensity of a mean pixel, R, G, B - color intensity of red, green and blue pixels appropriately). Results. Patients with diabetic foot syndrome had reduced proliferative activity of epidermal cells as they approach the sphacelous area of the affected limb, together with decreased resistance to apoptosis in the same areas. The share of cells positive for proliferating cell nuclear antigen was 58.74±4.8% in skin samples from the area of necrosis (n=6) compared to 77.41±3% (p <0.005) in skin samples of the healthy controls. The study of Bcl-2 marker expression by cells of the sphacelous area (n=6) revealed that mean greyscale intensity in patients with diabetic foot syndrome was 57.74±4.3, which was significantly lower compared to skin samples of the healthy controls (n=6, 89.69±3.4, p <0.005). Based on the expression of cytokeratin, it was shown that suprabasal layers of the epidermis cells were less differentiated in case of diabetic foot syndrome compared to the same cells of the epidermis of the control group. Conclusion. In advanced stages of diabetes, the combined effect of the disease is reduced number of epidermis proliferating cells, the reduction of their anti-apoptotic activity and loss of skin as an organ.

diabetes mellitus, skin regeneration, keratinocyte proliferation, apoptosis, angiopathy, immunohistochemistry, cell differentiation