Detailed descriptions of the methods are provided in Online Data Supplement at “http://hyper.ahajournals.org .”
In order to test whether the combination treatment of single elastase injection and pharmacologically-induced hypertension can cause the formation of intracranial aneurysms in mice, we treated C57BL/6J mice (8–10 weeks old) with a single stereotaxic injection of elastase at the right basal cistern, immediately followed by induction of systemic hypertension with continuous injection of angiotensin-II. After two weeks, we sacrificed the mice and perfused the animals with a bromophenol blue dye and gelatin mixture.
Our preliminary study indicated a wide variation in vessel diameter for the Circle of Willis and its major branches in mice. In order to have a conservative and consistent control for our experiment, we used the diameter of the basilar artery–one of the larger vessels in the brain circulation with relatively little variation—as a reference blood vessel. Aneurysms were operationally defined as a localized outward bulging of the vascular wall whose diameter is greater than 150% of the basilar artery diameter. The Circle of Willis or its major primary branches were assessed by two investigators in a blinded manner.
To assess the relationship between the elastase dose and the incidence of aneurysms, four groups of mice were prepared: Group 1: 35 milli-units of heat-inactivated elastase, Group 2: PBS (n=10), Group 3: 3.5 milli-units of elastase (n=10). Group 4: 17.5 milli-units of elastase (n=20), and Group 5: 35 milli-units of elastase (n = 44). Angiotensin-II was administered at 1000 ng/kg/min for two weeks in all groups.
To assess the relationship between the angiotensin-II concentration and the incidence of aneurysms, three groups of mice were prepared: Group 1: continuous infusion of PBS via osmotic pump (n=10), Group 2: 500 ng/kg/min of angiotensin-II (n=10), Group 3: 1000 ng/kg/min of angiotensin-II (n=44). This group (Group 3) is the same as the Group 5 in the above-mentioned experiment testing the dose dependency of elastase effects. 35 milli-units of elastase were administered to all groups.
In order to test whether the combination treatment of single elastase injection and pharmacologically-induced hypertension can cause the formation of intracranial aneurysms in mice, we treated C57BL/6J mice (8–10 weeks old) with a single stereotaxic injection of elastase at the right basal cistern, immediately followed by induction of systemic hypertension with continuous injection of angiotensin-II. After two weeks, we sacrificed the mice and perfused the animals with a bromophenol blue dye and gelatin mixture.
Our preliminary study indicated a wide variation in vessel diameter for the Circle of Willis and its major branches in mice. In order to have a conservative and consistent control for our experiment, we used the diameter of the basilar artery–one of the larger vessels in the brain circulation with relatively little variation—as a reference blood vessel. Aneurysms were operationally defined as a localized outward bulging of the vascular wall whose diameter is greater than 150% of the basilar artery diameter. The Circle of Willis or its major primary branches were assessed by two investigators in a blinded manner.
To assess the relationship between the elastase dose and the incidence of aneurysms, four groups of mice were prepared: Group 1: 35 milli-units of heat-inactivated elastase, Group 2: PBS (n=10), Group 3: 3.5 milli-units of elastase (n=10). Group 4: 17.5 milli-units of elastase (n=20), and Group 5: 35 milli-units of elastase (n = 44). Angiotensin-II was administered at 1000 ng/kg/min for two weeks in all groups.
To assess the relationship between the angiotensin-II concentration and the incidence of aneurysms, three groups of mice were prepared: Group 1: continuous infusion of PBS via osmotic pump (n=10), Group 2: 500 ng/kg/min of angiotensin-II (n=10), Group 3: 1000 ng/kg/min of angiotensin-II (n=44). This group (Group 3) is the same as the Group 5 in the above-mentioned experiment testing the dose dependency of elastase effects. 35 milli-units of elastase were administered to all groups.