40 Figure 2 Schematic diagram of stent deployment (A) and pro- cedure (B). (A): (1) The straightened nitinol wire stent inside the catheter, with pusher wire, ready for insertion into the animal. (2) At point of deployment, catheter is retracted over the pusher wire to deploy the stent. The nitinol wire re-coils using shape memory. (3) Fully deployed stent. (B) The femoral artery is exposed and ligated distal from the aorta. Blood ﬂow is tempor- arily stopped in the femoral using a sling and the stent catheter inserted through an arteriotomy. The catheter is pushed into the abdominal aorta before stent deployment. The catheter is then removed and the proximal femoral artery ligated. 48 h with aspirin (75 mg in 250 ml drinking water) and clopidogrel (25 mg/kg/day crushed and mixed with sardine). General anaesthesia was induced with isoﬂurane (maintenance rate of 1.5%) and analge- sic (0.03 mg/ml vetergesic, s.c.) given. The mouse was positioned with its left hind limb fully extended and as in line with its spine as possible so as to create a straight line between femoral artery and aorta. An incision was made in the groin, and the femoral artery and vein exposed by blunt dissection. The vessels were bathed in a solution of 1% lidocaine hydrochloride, and the femoral nerve dissected free of the artery before the femoral vein and artery were controlled proximally with slings and ligated distally with 6/0 silk ties (Figure 1B). Following arteriotomy (Figure 1C), the stent catheter was inserted into the femoral artery (Figure 1D and E) and slowly advanced into the abdominal aorta. Insertion into the aorta was performed without visual gui- dance. However, measurement of dissected specimens of similar weights showed that the renal arteries were just over 20 mm from the groin. Thus, the stent catheter was inserted to a distance of 20 mm. The stent was deployed and the catheter withdrawn before ligation of the femoral artery proximal to the arteriotomy, the wound closed, and the animals allowed to recover. Double anti- platelet therapy was continued post-operatively until harvest. As a control, sham-stent procedures (n ¼ 6) were also performed, following the stent protocol but without deployment of a stent. J. Chamberlain et al. 2.4 Tissue processing and histological analysis Animals (n ¼ 8 – 12 per timepoint) were killed and perfusion ﬁxed 1 h, 1, 3, 7, and 28 days post-stenting. Stented sections were embedded in glycol methacrylate resin (Technovit 8100, TAAB laboratories) or modiﬁed Wolf methacrylate resin8 (refer Supplementary material online) and transverse sections, 10 mm thick, made using an Isomet 5000 diamond tipped saw and Metaserv 2000 grinder/polisher (Buehler). Morphometric analysis was performed on H&E and EVG stained sections, using Lucia digital analysis software. Each aorta generated two to three sections of sufﬁcient quality for analysis, all of which were used in neointimal area analysis. 2.5 Immunohistochemical analysis Immunohistochemical analysis was performed to determine endo- thelial cells (vWF, Dako, UK), inﬂammatory cells (mac-387, Dako), and smooth muscle cells (a-smooth muscle actin, Dako). Slides embedded in modiﬁed Wolf methylmethacrylate were deacrylated in xylene and 2-methoxyethylacetate, followed by rehydration in acetone and water, or rehydrated in ethanol, followed by a water wash for sections embedded in T8100 resin, prior to staining. Antigen retrieval, by heating to 808C for 20 min in citrate buffer, pH6, was performed for vWF and mac-387 staining. Sections were incubated with primary antibody for 3 h at 378C in PBS containing 10% normal serum. Positive staining was visualized using the ABC tech- nique followed by New Fuschin. Sections were dry mounted using superglue, without counterstaining. Further details on antibody dilutions and incubation times for each stain are given in Supplemen- tary material online, Table S1. 2.6 Statistics Data are presented as mean + SEM. Timecourse data were analysed using a one-way ANOVA followed by a Bonferroni post test. A Stu- dent’s t-test was used for comparison of IL-1R12/2 vs. IL-1R1þ/þ neointima formation. P , 0.05 was considered signiﬁcant. 3. Results Several prototypes of stent were designed, developed, and tested. The ﬁnal design was a coil with external diameter 1.0 mm, length 3.0 mm, and a low pitch; the latter reducing the number of rotations made by the stent during deployment. As the stent exits the catheter eccentrically and caused perforation of the vessel with initial prototypes, the tip was blunted with a laser welded bead, and a 1.0 mm straight ‘leg’ added to act as a stabilizing strut lying against the vessel wall during deployment, giving the stent a ﬁnal length of 4.0 mm. The stent was developed and tested in 42 animals. The ﬁnal design was then deployed in 76 mice, with 69 resulting in successful recovery of a stented aorta. All arteries were patent upon harvest. The 9% of animals that failed were lost due to procedural pro- blems. These involved failure of the stent to deploy from the cath- eter (four animals), failure to insert the catheter into the arteriotomy (two animals), and stent migration into the abdominal cavity (one animal). The stent procedure can be undertaken with conﬁdence after training on approximately six mice and takes 30 min to complete, in the hands of an experienced operator, with minimal blood loss. Excessive blood loss is controlled by slings providing a temporary