Alzet 2004

Beginning at 8 wk of age, transthoracic ECG was obtained as above. After anesthetizing the animal with subcutaneous injection of ketamine/xylazine (90 and 4.5 mg/kg, respectively), a subcutaneous osmotic pump (Alzet 2004; Durect, Cupertino, CA), containing either vehicle or erlotinib (5 mg/ml), was placed in the subcutaneous tissue immediately lateral to the spine on the posterior of the animal. After 4 wk, the initially placed pump was removed and replaced with a second pump containing the same treatment (vehicle vs. erlotinib). A follow-up transthoracic ECG was obtained after 8 wk of treatment to assess aortic root diameter, as detailed above.

Osmotic pumps (Alzet 2004; Durect, Cupertino, CA, USA) containing angiotensin II (AngII, 1000 ng/kg/min, Sigma-Aldrich) were placed subcutaneously into 8–12-week-old ApoE^−/− male mice, as previously described^{46 (link),62 (link)}. All mice were fed unlimited water and placed on a high-fat diet (TD 88137, Harlan Teklad, Indianapolis, IN, USA) with no restrictions on movement. A separate group of mice were treated with intraperitoneal injections of PBN from day 1 to day 27. Aortic diameters in all groups were measured and aortic tissue was harvested on day 28 for further analyses.

For the diameter study 47 male ApoE^-/- mice on a C57Bl/6J background were purchased at the age of 20 weeks from Janvier (Saint Berthevin, France). All animals were scanned with ultrasound at baseline (day 0). The animals were subsequently implanted a 200 μl osmotic pump (model Alzet 2004; Durect Corp, Cupertino, CA), filled with Angiotensin II or a combination of Angiotensin II with either Angiotensin-(1–7) or (D-Ala⁷)-Angiotensin-(1–7) (all of them purchased at Bachem AG, Bubendorf, Switzerland) diluted in sterile saline 0.9%. Animals were subdivided into 3 groups to study the effect of the aforementioned peptides on aneurysm incidence and phenotype Nevertheless, all animals developed similar aneurysm phenotypes with no significant differences between groups. As such, data were pooled for the purpose of this methodological study. Eleven animals died from aneurysm rupture in the first 14 days, and 1 additional animal died in the last 14 days. Necropsy was performed to confirm hemoabdomen or hemothorax as the cause of death. A rupture rate of 20% within the first weeks of angiotensin II infusion is in agreement with previously published values [22 (link), 45 (link), 46 (link)]. Hence 34 animals were scanned with ultrasound 14 days after pump implantation, and 33 animals were scanned a third time after 28 days.