Cardiovascular Journal of Africa: Vol 32 No 5 (SEPTEMBER/OCTOBER 2021)

CARDIOVASCULAR JOURNAL OF AFRICA • Volume 32, No 5, September/October 2021 AFRICA 257 In the control group, no clamp or tourniquet was used. A longutidinal incision was made on the anterolateral side of the crus muscle. A 5 cm length of tibialis anterior muscle was excised. A longitudinal anteromedial incision was made over the femoral neurovascular bundle. The neurovascular bundle and rectus femoris muscle were marked with 4.0 vicryl suture rope from the proximal and distal borders. Then the bundle and rectus femoris muscle were excised. Histological examination of the common femoral artery, vein, nerve, rectus femoris and tibialis anterior muscle was performed (Figs 1, 2). The animals were euthanised. Areas with significant histological findings were evaluated. In the clamp group, the arterial specimen was divided into three groups: proximal, the clamp area in the middle, and distal. The aim of this part of the study was to compare the normal tissue in the proximal part of the ischaemic area, the damage to the arterial tissue where the clamp was applied, and the changes due to ischaemia in the distal part of the clamp. In the tourniquet group, the artery sample was divided into three similar pieces. The aim of this part of the study was to evaluate the effect of pressure differences on the tissue between the proximal and the middle part of the tourniquet cuff, and to examine the effect of ischaemia on the distal side. In both the tourniquet and the clamp groups, the vein, nerve and muscle tissue were divided into three sections: proximal, middle and distal. In the clamp group, the aim was to evaluate the tissue injury caused by dissection. In the tourniquet group, the aim was to evaluate the effect of pressure difference between the proximal and middle part of the tourniquet cuff, and to examine the effect of ischaemia on the distal side. The tibialis anterior muscle tissue was examined in all three groups to compare the ischaemic injury distal to the extremity. Connective and adipose tissue were not evaluated because no significant light microscopic findings were expected with short-term trauma and hypoxia in these kinds of tissues. Six slides for the vessels and three slides for the nerves and muscles were obtained per sample. Samples from each group were fixed with 10% neutral-buffered formalin for one day and processed for standard paraffin embedding. Serial sections (4-µm-thick slices) were cut using a microtome. All of the sections were stained with haematoxylin and eosin (H&E) and vessel sections with elastin vanGieson stain (Ventana) for light microscopic examination (Nikon-Eclipse-80i-DS-Ri1). An automatic device (Ventana, Benchmark XT) was used for histochemical staining. The photographs were captured with a digital camera (Nikon-Eclipse-80i-DS-Ri1). The pathologist, who was blinded to the slide numbers and groups, examined the slides for tissue damage, including sections from the tibialis anterior and rectus femoris muscles, the femoral nerve and the femoral artery and vein. Degeneration and inflammation were evaluated semi- quantitatively for scoring skeletal muscle injury. Histological findings such as cytoplasmic eosinophilia with loss of cytoplasmic structures, cytoplasmic vacuolation, swelling, loss of striation, fragmantation and rupture were used for scoring muscle degeneration. The score was: 1, findings are mild and focal; 2, moderate and in some areas; 3, severe and common. The following criteria were used for inflammation: score 0, no inflammation; 1, mild inflammation; 2, moderate inflammation, and 3, severe inflammation. The scores were added and the total muscle injury scores were calculated for each group. Degenerative changes in the peripheral nerve fibres were determined semi-quantitatively according to oedema and axonal degeneration using light microscopy. If the findings were mild, the score was 1; moderate, 2; and severe, 3. Light microscopy gives limited information without electron microscopy findings. Essentially we did not expect serious damage to the nerve as the clamp or tourniquet was applied for a short time only. Arterial injury was scored using the criteria in Table 1. Endothelial injury was scored using the same method applied for arteries and venules. Venous intimal plaque was also evaluated. Using a quantitative approach, intact endothelium in the 500-µm segment was assessed by number of endothelial cells (NEC) in the arteries and venules. Endothelial damage (score of endothelial ınjury: SEI) was assessed in the venules and arteries. The continuity of the internal elastic lamina (score of lamina elastica interna injury: SLEI) in the arteries was examined, as well as the smooth muscle vacuolation in the medial layer (tunica media smooth muscle vacuolation: TMSMV) in the arteries. Western blot analysis was used for protein analysis. Tissue samples from the distal part of the clamp group ( n = 7), the distal part of the tourniquet group ( n = 7) and the control group ( n = 7) were snap frozen in liquid nitrogen and stored at –80°C. Being a key regulator for tissue injury TNF- α and also for loading control, beta-actin antibodies were used for analysis and bands were determined using the imaging system (Vilber Fusıon FX, France). Statistical analysis Power analysis was done before the experimental set up to determine the number of animals required. The difference between the groups was predicted using Margovsky et al . ‘area of endothelial damage’ values. 9 The effect size ( d ) was 2.19 in the calculation for obtaining 80% power at the α = 0.05 level. Accordingly, it was determined that there should be at least seven members in each group. Descriptive statistics were used to define continuous variables (mean, standard deviation, minimum, median, maximum). Comparisons of independent variables with normal distribution were performed using the Student’s t -test. Comparisons of two independent and non-normal distributions were performed using the Mann–Whitney U -test. The chi-squared test (or Fisher’s exact test at appropriate locations) was used to examine the relationship between categorical variables. The statistical significance level was determined as 0.05. The analysis was performed using MedCalc Statistical Software version 12.7.7 Table 1. Score parameters of vessel injury. Total score: 0–3: mild injury, 4–6: moderate injury, 7–9: severe injury Endothelial damage score TMSMV None 1 None 0 Minimal 2 Some cells in deep 1 Moderate desquamation in endothelial cells 3 Consistent 2 Significant desquamation in endothelial cells 4 Superficial + deep 3 Continuity of internal elastic lamina Inflammation score Intact 0 None 0 Interruption/fissure 1 Mild 1 Significant fissure 2 Moderate 2 Significant fissure/cavity + thrombus 3 Severe 3 TMSMV: tunica media smooth muscle vacuolation.