Scattering of inflammatory cells across diabetic tissue and infiltration of inflammation from adventitia to intima vessels
According to the results, inflammatory cells were observed in the whole diabetic tissue. However, a significant increase in the number of inflammatory cells was seen in the vascular wall compared to the intima (P < 0.0001)(Fig. 1).
IL-1 was increased in the wound area and vascular adventitia
The increased cytokines IL-1 occur in the majority, if not the whole, conditions of inflammation and have been acknowledged to be targeted by therapeutical interventions12. According to the results, the amount of IL-1 in the diabetic ulcer area was significantly higher than the central area of the tissue (P < 0.0001), which according to the immunohistological results of IL-1 showed the increasing trend of inflammation from the wound side into the tissue. Examination of the vessel wall also showed an increase in IL-1 in the Adventitia compared to the intima (P < 0.01) which was increasing from the adventitia to the intima.
IL-6 was increased in diabetic tissue and vascular adventitia layer
The increased cytokines IL-6 occur in the majority, if not the whole, conditions of inflammation and have been acknowledged to be targeted by therapeutically interventions13. According to the results, there were high levels of IL-6 throughout the diabetic tissue, which was more common in the ulcer area (P < 0.05). In the vascular wall, the amount of IL in the adventitia was significantly higher than in the intima (P < 0.0001)) Fig. 3).
PECAM-1 was increased in the Ulcer area and the vascular adventitia
Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a molecule the expression of which occurs on the entire cells inside the vascular chamber. It shows varying expression levels on the majority of leukocyte subtypes, platelets, and endothelial cells14.
According to the results of immunohistochemically staining, the level of PDEA protein in the wound area and around the wound is high, which is significant higher that the ulcer area (P < 0.01). According to the pathological slides, it can be interpreted that the inflammation gradually spreads from the Adventitia layer to the intima layer (P < 0.05) (Fig. 4).
VECAM levels were increased in the ulcer area and the vascular adventitia layer
VCAM-1 was initially recognized as a cell adhering molecule helping in the regulation of inflammation-linked vascular adherence and the transendothelial relocation of leukocytes, including macrophages and T cells. Recently published reports are suggestive of a close association of VCAM-1 with the progress of several immunological ailments15. According to immunohistochemically staining, the expression of VECAM protein was significantly increased in the wound area compared to the extra wound area and the vascular Adventitia layer compared to the vascular intima layer(P < 0.0001).
HNE (oxidative stress) levels were high in the wound area and intravascular layer
High HNE levels have been associated with some sorts of diseases that involve redox imbalance: cardiovascular diseases, macular degeneration, metabolic syndrome, cancers, and atherosclerosis. In the mentioned diseases, not only HNE is a simple oxidative stress marker.
HNE-4 levels in the wound area increased significantly compared to the extra-wound area, which showed that it was spreading from the wound area to the surrounding tissues (P < 0.0001). HNE-4 levels were higher in the vascular intima compared to the adventitia layer (P < 0.01)) Fig. 6).
Disruption of elastin fibers and accumulation of collagen fibers in the vascular layer were observed
According to elastin staining, rupture of elastin fibers was seen in inflamed tissue areas such as the wound area and the vascular adventitia layer. If there were signs of collagen fiber accumulation in the vascular wall and throughout the diabetic tissues, including ulcers)Fig. 7).