2.1 Animal preparation
All animal experiments were conducted in accordance with the Guide for
the Care and Use of Laboratory Animals of the US National Institutes of
Health, the Animal Experiment Regulations of the University of Tsukuba,
and the Basic Guidelines for the Proper Conduct of Animal Experiments in
Academic Research Institutions under the jurisdiction of the Ministry of
Education, Culture, Sports, Science, and Technology of Japan. Animal
experiments were performed with approval from the University of Tsukuba
Animal Experiment Committee.
Male C57BL/6 mice (eight weeks old; Japan Charles River Kanagawa, Japan)
were randomly assigned to three groups: control, AngII-infused (AngII),
and AngII and isorhamnetin-treated (AngII+isorhamnetin). AngII (1,000
ng/kg/h) was administered continuously for two weeks using an osmotic
minipump (Alzet, model 2002; Cupertino, United States). Normal saline
was injected into the mini-osmotic pump in the control group.
Isorhamnetin (5 mg/kg) was intraperitoneally administered daily for
three weeks to mice in the AngII+isorhamnetin group one week prior to
AngII administration. Isorhamnetin was suspended in 0.1% dimethyl
sulfoxide (DMSO) and 1% polypropylene glycol. In addition, the same
amount of vehicle solution (0.1% dimethyl sulfoxide, 1% polypropylene
glycol, and saline) was administered intraperitoneally as a sham
injection to the mice in the control and AngII groups. After three
weeks, the mice were euthanized by CO2 inhalation and
their hearts were harvested for further experiments. The mice used to
obtain biochemical and histological data were not assigned to the AF
induction study to avoid causing mechanical effects associated with
catheters.