Surgical aspects
All saphenous veins were examined by echography before surgery to determine their size, morphology and functionality. Veins with any abnormalies, such as regurgitation, varix or varicose conditions, were excluded from graft candidates. CABG was performed in an on-pump or off-pump fashion by the surgeon’s choice. There were no marked differences in the handling of SVGs between on- and off-pump procedures. In on-pump cases, the surgical procedure was performed under cardiopulmonary bypass with moderate hypothermia (28 to 30 ℃) and cardiac arrest with tepid blood cardioplegia. Vein grafts were mainly bypassed to the right coronary artery (RCA) and left circumflex artery (LCX), and the left anterior descending artery was reconstructed by the internal mammary artery. Vein grafts were anastomosed to the RCA or LCX, followed by perfusion with tepid blood cardioplegia (on-pump) or blood (off-pump) and anastomosis of the other side to the aorta.
SV harvesting techniques
1) CV group
The SV was exposed mainly from the lower leg by skipped longitudinal leg skin incisions, wherein the side branches were ligated. The vein was removed from the leg after dissection, connected to the cannula inserted into the femoral artery and dilated with arterial pressure for 10 min with blood mixed solution before the sewing of the anastomoses. The residual part of the vein was cut off from the central side (aortic side) after the anastomosis of the distal side to the RCA or LCX. In CV group, the vein was manually distended with blood mixed solution (30 ml blood, 3000 U of heparin sodium and 30 mg of papaverine hydrochloride) at <300 mmHg. The adventitia was stripped off [16]. The harvested vein was stored in wet gauze at room temperature in an operating room. A few hours later, the stored vein was washed with PBS and fixed with specific buffer in our laboratory.
2) NT group
The SV was exposed via the same skin incisions as used for CV. The vein was isolated along with 5 mm of the surrounding fat tissue, and all visible side branches were ligated. After removal, the vein was connected to the cannula inserted into the femoral artery and dilated with arterial pressure for 10 min with blood mixed solution before the sewing of the anastomoses. The residual part of the vein was obtained as in the CV group. In the NT group, the vein was neither flushed nor distended manually. The adventitia was not removed. The harvested vein was stored and treated as in the CV group thereafter.
SVG pre-bypass samples (from Patient No. 1- No. 5)
The residual SVG samples were obtained from Patient No. 1 via CV, Patient No. 2 via NT, and Patient No. 3- No. 5 via both CV and NT. The endothelial surface of SVGs from CV group (No. 1 and No. 3) and NT group (No. 2 and No. 3) was analyzed with SEM. The endothelial integrity of SVGs from CV group (No. 1, No. 4 and No. 5) and NT group (No. 2, No.4 and No. 5) was analyzed with immunohistochemistry.
SVG autopsy samples (from Patient No. 6- No. 7)
1) Patient No. 6
The patient was a 75-year-old male. He had suffered acute myocardial infarction of the inferior wall. CAG revealed three-vessel disease, and he received CABG (on-pump, arrested heart) with LITA to LAD, SVGs to D1, 14PL, 4PD and 4PL. The CV technique was used to harvest SVGs. He ultimately died on day 8 after surgery due to low-output syndrome. The grafted SVG was harvested at the autopsy. Sample was prepared from the SVG to 14PL
2) Patient No. 7
The patient was a 78-year-old male. He had suffered unstable angina. CAG revealed three-vessel disease, and he received off-pump CABG with RITA to LAD, LITA to 14PL, SVGs to D1, D2, 4PD and 4PL. The NT technique was used to harvest SVGs. He ultimately died on day 7 after surgery due to massive pulmonary embolism. The grafted SVG was harvested at the autopsy. Sample was prepared from the SVG to D1 and D2.
The patients’ characteristics are summarized below.
Patient No.
|
Age
|
Sex
|
On-/Off-
pump
|
Preparation
|
Analysis
|
1
|
71
|
M
|
On
|
CV
|
SEM and immunohistochemistry
|
2
|
73
|
F
|
Off
|
NT
|
SEM and immunohistochemistry
|
3
|
77
|
M
|
On
|
CV and NT
|
SEM
|
4
|
78
|
M
|
Off
|
CV and NT
|
immunohistochemistry
|
5
|
68
|
M
|
On
|
CV and NT
|
immunohistochemistry
|
6
(autopsy)
|
75
|
M
|
On
|
CV
|
immunohistochemistry
|
7
(autopsy)
|
78
|
M
|
Off
|
NT
|
immunohistochemistry
|
SEM
The SEM analysis was conducted as described previously [17]. A few hours after resection, the SVG was fixed with 2% glutaraldehyde. We commissioned the scans and analyses from Hanaichi Ultrastructure Research Institute (Okazaki, Japan). The SVG section was washed, postfixed, dehydrated and examined by SEM (JSM-7500F; JEOL, Tokyo, Japan). We received the examination report from the company and also analyzed the images by ourselves.
Immunohistochemistry
Immunohistochemistry was conducted as described previously [18]. A few hours after resection, the SVG was fixed with 10% formalin overnight at room temperature and embedded in paraffin. Tissue sections (3 µm) were stained with anti-von Willebrand Factor antibody (#65707; Cell Signaling Technology, Beverly, MA, U.S.A.). All images were captured by a BZ-X710 microscope (Keyence, Osaka, Japan) and analyzed using the Image J software program (National Institutes of Health).