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sections which are then incubated in TTC for determination of
infarct size and the area at risk (AAR is stained in the red
color).
3. Hearts are cut into two to three transverse sections (perpen-
dicular to the long axis) starting from slightly above the coro-
nary ligature (in case of permanent ischemia the thread is in
place and visible), or above the visible area of injury (in case of
ischemia-reperfusion the thread is preferably not left in place
to decrease the occurrence of adhesions between epicardium
and chest wall). The sections are put in 10% buffered formalin
to be processed within 1 month for paraffin embedding.
Methods 1 and 2 are usually employed in case of long-term
experiments (>7 days follow-up), while method 2 is for short-term
experiments (2-24 h).
Myocardial tissue sections are subjected to Masson's trichrome
and hematoxylin-eosin staining. Myocyte cell size and density are
measured in H&E-stained sections (see Note 3).
To avoid likely biases, it is crucial that the person assessing all
histological slides is blinded to a grouping.
4
Notes
1. Fractional shortening (FS) is calculated from the composite
LV internal, diastolic (LVIDd) and systolic (LVIDs) dimen-
sions as: FS = ((LVIDd − LVIDs)/LVIDd) × 100 from
M-mode short axis view. Left ventricular (LV) volumes and
LVEF are calculated by the modified simple plane Simpson's
rule from the parasternal long-axis view. The MI size at the
mid-papillary muscle level is estimated from 2D short axis
LV images and expressed as a percent of the LV endocardial
circumference. Infarct area is identified as a sharply demar-
cated section of the LV free wall that fails to thicken during
systole. The length of the akinetic part of the LV endocardial
circumference is measured from freeze-frame images at end-
diastole. Aortic outflow and diastolic transmitral LV inflow
velocities are measured from 4 to 5 apical chamber views
respectively by pulsed-wave Doppler with a sample volume
length of 3.5-7.5 mm; the ultrasound beam is aligned as par-
allel as possible to Color Doppler flow and to record the
highest velocities.
2. Usually the anesthetized rat/mouse is placed in a horizontal
bore 7 T Biospec preclinical scanner with a shielded gradient
insert (BGA 12, 40 mT/m; rise time, 110 ms). A quadrature
or linear volume coil and rat/mouse surface coil are used to
transmit/receive the magnetic resonance signals (Quadrature
or Linear volume coil inner diameter, 72 mm; rat/mouse
surface coil inner diameter, 20 mm/10 mm). After localization
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