Statement
All experiments and methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by a named institutional/licensing committee. Specifically, animal research protocol was approved by Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital. The study was carried out in compliance with the ARRIVE guidelines. All animals were euthanized with an intravenous injection of a lethal amount (7-10 ml) of xylazine hydrochloride under deep anesthesia.
Animal Model Preparation
Thirty New Zealand white male rabbits weighting 3000-3500 g were used in our study. All animals were housed in cages with a 12-hour, light/dark cycle and ad libitum access to water. For anesthesia, 5 mg/kg body weight tiletamine-zolazepam (Zoletil 50; Virbac, Carros, France) and 2 mg/kg body weight of 2% xylazine hydrochloride (Rompun; Bayer, Seoul, Republic of Korea) were injected intramuscularly in the posterior thigh. Atherosclerosis was induced as described in previous studies 18-22. 10 rabbits in the atherosclerosis group were fed an atherogenic diet consisting of 0.5% cholesterol and 6% peanut oil for 5 weeks and then switched to a diet containing lower cholesterol (0.025%) for another 5 weeks to prevent liver failure 18,22. The 20 rabbits in the control group (group B) were fed normal rabbit chow for all study periods. Study protocol for induction of atherosclerosis is schematically shown in Figure 1.
Angiography
All rabbits underwent angiography to evaluate the presence of significant stenosis or occlusion of bilateral aortoiliac and femorotibial arteries, one day prior to CT scan. After sedation, rabbits were placed in supine position and right central ear artery of each rabbit was cannulated using 16 gauze IV cannula. To access the infrarenal aorta, a 2.0-Fr microcatheter (progreat a, Terumo, Tokyo, Japan) was advanced via the ear artery to the iliofemoral artery and femorotibial angiography was performed.
Foot Perfusion CT
Perfusion CT protocol similar to previous experiment was performed to evaluate foot perfusion status of each rabbit as detailed in a previous study 1717. In brief, each animal was laid down on the table of a 64-detector CT scanner (Ingenuity; Philips Medical, Einthoven, the Netherlands) with 4-cm z-axis coverage in volume scan mode. First, baseline perfusion CT scan was obtained by using following parameters: scan coverage including the whole hindfoot and ankle, volume scan mode without table movement, gantry rotation time of 400 msec, detector collimation of 64 x 0.625 mm, tube voltage of 100 kV, and tube current of 100 mA. The dynamic volume scanning started 1–3 seconds after intravenous bolus injection of 3mL of iopamidol (Pamiray, 370mg of iodine per milliliter; Dongkook Pharmaceutical, Seoul, South Korea) contrast material. Contrast material injection was followed by injection of an equal volume of normal saline, both injected with an automated dual-rail injector (Stellant; Medrad, Warrendale, Pa) at a rate of 1 mL/sec via the left auricular vein. After 5 minutes of rest, a disposable neonatal blood pressure cuff (Welch Allyn, NY, USA) was applied to animal’s left thigh and cuff was inflated to 200 mmHg to block blood flow to left foot for 3 minutes. As the cuff deflated to 0 mmHg within 3 seconds, a second perfusion CT was obtained in the same manner as the previous scan 23-26. Perfusion CT protocol timeline was illustrated in Figure 1.
Image reconstruction and analysis of perfusion CT findings
All the CT images were transferred to an image archiving and communications workstation (INFINITT; Infinitt healthcare, Seoul, Republic of Korea) and analyzed by in-house perfusion analysis software 17. The image reconstruction method for the perfusion CT data was identical to that of a prior animal experiment 17, except that the plantar dermis in the heel base rather than that of the toes was used as the representative tissue to set the upslope time range. Each session of perfusion CT data was sent to the software and a pixel-based, color-coded perfusion map was generated by using the upslope method 17. Based on a perfusion map, region of interest (ROI) for posterior tibial artery and plantar dermis was placed, and time-attenuation curve 1 was calculated respectively (Fig 1). Upslope time range was set from the arterial time-attenuation curve and the following perfusion parameters were obtained; Peak arterial enhancement (PAE), Time to peak enhancement (TTP), and maximal upslope (m). Time attenuation curve of each pixel was normalized by the maximal arterial enhancement value. Quantitative analysis of perfusion parameters was performed by two radiologists (S.H and H.D.M) in consensus. Regional blood flow at baseline perfusion CT scan was measured at the dermis of the foot sole by placing an ROI on the axial image. In the same way, ROI was set in the corresponding location of the plantar dermis on the perfusion map obtained from the second CT scan, and each perfusion value and the ratio before and after inducing CRH was calculated.
Pathological analysis
All animals were preanesthesized and sacrificed with an intravenous injection of xylazine hydrochloride. Dermal tissues of left heel of all rabbits were harvested. Tissues obtained were weighed and half of the tissues were processed to evaluate number of each color’s microsphere, the other specimens were fixed in 10 % buffered formalin and were embedded in paraffin. Thereafter, tissue samples were treated with hematoxylin and eosin staining for basic histopathological examinations and consecutive section was treated CD 31 staining (DAKO Corp., Carpinateria, CA, USA) for microvascular density (MVD) evaluation. After digital images of the histologic slides were obtained (Leica Microsystems, Mannheim, Germany), MVD was calculated using image analysis software (Image J, version 1.45s; National Institutes of Health, Bethesda, MD) (11). The five hot spots with the most intense vascularization were selected by L.J.H who was blinded to information of animal group, while screening at a low-power field (×40). MVD counts of the five areas per the hot spots were performed at a high-power field (S.B.C) (×100). Any brown-stained endothelial cells or endothelial cell clusters clearly separated from adjacent microvessels were counted as one microvessel stained by the anti-CD31 antibody, irrespective of the presence of a vessel lumen. The mean microvessel area percentage of all the measured areas was determined to be the MVD.
Statistical analysis
All data in the study was reported as mean ± SD. The perfusion value and ratio of regional blood flow between baseline and CRH in each group was tested by using paired-T test and Wilcoxon signed rank test. The value of change of each parameter between two groups were compared using repeated measures analysis of variance test. The data processing and analysis were performed using Statistical Package for the Social Sciences version 18.0 (SPSS Inc, IBM Company, Chicago, IL). A two-sided P value of less than 0.05 indicates that the groups differ significantly in terms of statistical results.