Male ApoE−/− mice, approximately 8 weeks of age, were immunized subcutaneously on days 0, 14, 21, 28, 56, 94, and 122 or 0, 14, 28, 56, 84, 112, and 140 with 50 μg of IL-1α-C-Qβ (n = 13) or Qβ-VLP (n = 12 or 10). Mice were fed a normal chow between day 0 and 20, and a western diet (20% fat, 0.15% cholesterol, no cholate, Provimi Kliba AG, Switzerland, or 21.2% fat, 0.2% cholesterol, Harlan, Netherlands) from day 21 to the end of the experiment. Mice were bled at regular intervals throughout the experiment and the antibody response against IL-1α was measured in plasma. Sacrifice was on day 159, and the aorta were isolated and prepared essentially as described . The animals were bled by cardiac puncture and perfused with cold PBS. The aorta was sectioned 2 mm from the heart and further processed. In addition, hearts were removed and snap-frozen in liquid nitrogen for subsequent histologic preparation. The aortic arch was sectioned 5 mm down from the left subclavian artery and immediately frozen in liquid nitrogen. The heart was sectioned in the middle, and the upper part was immediately frozen in Hank's balanced salt solution in a plastic tube in liquid nitrogen for histological analysis essentially as described [33, 34]. The serial sections (7 μm thickness) were cut through the origin of the aorta and harvested upon appearance of at least two valve cusps, until disappearance of the last valve cusps. Sections were fixed in formalin, stained with oil red O, and plaque load was evaluated in 4–7 sections per mouse (for one animal of the Qβ group only three sections were available) by quantitative image analysis (Image J). An average plaque area was computed for each animal, and an average group plaque area was computed for the IL-1α-C-Qβ and Qβ group, respectively. Statistical analysis was performed with a Student's t-test.
For the evaluation of atherosclerosis in the descending aorta, these were further cleaned from residual adventitia on a glass petri dish filled with cold PBS. The aorta were cut longitudinally, pinned out on a black wax surface, and fixed overnight in 4% formalin. They were then stained overnight in oil red O. The plaques were quantified with imaging software on digital photographs. The plaque load was expressed as the sum of the surface of all plaques of the aorta taken up to the iliac bifurcation, divided by the total surface of the aorta measured up to the iliac bifurcation, in percentage. The difference in median plaque load between the IL-1α-C-Qβ and Qβ groups was analyzed using a Mann–Whitney test.
Inflammatory infiltrates in the adventitia were scored by a pathologist, using a scoring scheme ranging from 0 to 4 in 0.5 increments. Two rounds of scoring were performed, each with the sections blinded and ordered in a different sequence. Three to four sections per animal were quantified, with the exception of one animal in each group where only two sections were available. An average score was computed, and the data analyzed with a Mann–Whitney test.
CD-68 staining on cryosections was performed by shortly fixing the sections in formalin. Peroxidase activity was blocked by incubation with H2O2. The CD68-positive area was quantified in the intima and media of three to four sections per animal (for two animals of the IL-1α-C-Qβ group two sections only were available). Average plaque area was computed and statistical analysis performed with a Student's t-test.