Assessment of Intracellular Calcium in Cerebral Artery Myocytes - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 531

that includes EGTA (5 mM), ionomycin (10 μ M), and nigericin

(5 M). Recording at the end of this period allows determination

of R min . EGTA is then washed from the bath by perfusion of base-
line solution for 15 min. To obtain R max , arteries are then perfused
(15 min) with baseline solution containing CaCl 2 (10 mM), iono-
mycin (10

μ

M) for 10 min. The ratio of
calcium-free over calcium-bound fl uorescence intensities at F 380 (

μ M), and nigericin (5 μ

)
is determined from these values. In our studies, we have used an
apparent dissociation constant ( K d ) of 282 nM of fura-2 for Ca 2+
determined by Knot and Nelson ( 2 ) using rat cerebral arteries.

β

Luminal diameter is simultaneously recorded throughout the entire
experiment using a CCD camera and the length-calibrated video
edge detection function of IonOptix software (Figs. 1d and 2 ).
Constriction is analyzed as the decrease in arterial diameter relative
to the passive diameter using the following equation ( 22 ):

2.3.3. Measurement
of Arterial Diameter

DP − DA

%Constriction = × 100,

DP

where DP is the passive (fully dilated) diameter of the artery in
Ca 2+ -free aCSF containing a VDCC blocker (e.g., 100 M diltiazem

or 300 nM nifedipine) and a second vasodilator, such as forskolin
(1

μ

M) and DA is the active diameter of the artery in response to
the stimulus in aCSF. Percent dilation can be obtained using the
following formula ( 22 ):

μ

DV − DA

%Dilation = × 100,

DP − DA

where DV is the arterial diameter in the presence of vasodilator,
DA is the active arterial diameter before application of vasodilator,
and DP is the passive arterial diameter.

Most investigators obtain ratiometric measurements of fura-2 in
isolated arterial segments or dissociated cerebral artery myocytes
by collecting emitted photons using a PMT. Fluorescence detec-
tion may also be achieved using electron multiplying CCD or
intensifi ed CCD cameras along with quantitative ratio imaging
software for analysis and estimation of intracellular Ca 2+ ( 2 ).
However, high sensitivity cameras required for these measurements
are expensive and do not offer signifi cant spatial information when
used with wide-fi eld microscopy. In cases where an investigator
wishes to measure fura-2 fl uorescence in a specifi c layer of the vas-
cular wall (smooth muscle or endothelium), fl uorescent imaging
over photometric methods may be advantageous.

2.3.4. Variations in
Measurement of Global
[Ca 2+ ] i