2.4. Immunohistochemistry Protocol
Histopathological changes observed in vein tissue samples, conducted by three harvested techniques, were graded as follows: Grade 0—without any structural damage to the entire vein wall; disturbances, defined as grade 1, included minimal damage to the endothelial integrity with a discreet stretch of the detached intimal layer (<10%); it also included a trace defection of the perivascular fat and the connective tissue of the adventitial layer, without structural injury to the medial layer and vasa vasorum. Grade 2 included focal endothelial stretching or detaching (<25%), without damaging the intern elastic membrane and medial layer, but with mild damage to the adventitial connective tissue and focally detached vasa vasorum; this included moderate damage to the perivascular fat tissue. Grade 3 was based on moderate endothelial stretching or stripping (25–50%), in some cases focally detached, with minimal damage to the intern elastic membrane and medial layer, but with moderate damage to the adventitial tissue and vasa vasorum. Grade 4 was marked for tissue samples that showed a partially detached endothelium (>50%), damaged intimal elastic membrane, some focal areas of separated smooth muscle fibers of the medial layer, and a damaged adventitial layer with a partially absent vasa vasorum; this was also without the presence of the perivascular fat. Grade 5 included very marked changes to the luminal integrity, with a mostly stripped endothelium (>75%) and a segmentally separated intimal layer from the deeper layers; it also included damage to the intimal elastic membrane with the discontinued elastic and smooth muscle fibers of the medial layer, and extensive damage to the adventitial layer and vasa vasorum without the surrounding perivascular adipose tissue. Grade 6 was defined as vein tissue samples that had shown extensive structural damage to all three layers; this included a detached endothelial and medial layer, a stripped adventitial layer, and the absence of the vasa vasorum.Immunohistochemical analysis was performed on routinely prepared 4 µm thick vein sections. The wall integrity of the saphenous veins was estimated using the antibodies against CD31, Factor VIII, and Caveolin, while the endothelial function was evaluated using endothelial nitric oxide synthase (eNOS). All tissue slides were examined and scored as follows: Grade 0—no staining, Grade 1—trace, Grade 2—minimal, Grade 3—moderate, Grade 4—strong.The entire protocol of pathohistological examination is presented in Methods IV in the The main aim of the study was to analyzing the influence of three different surgical techniques on vein structural damage.