Spreading Depolarization - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 328

Fig. 2. Imaging of SD using IOS in the acute mouse brain slice. Pseudocolor overlay
represents relative changes in light transmission. Two ion-selective/reference electrodes
are placed in a midcortical layer.

scientifi c-grade CCD microscope camera, which usually features
cooling for noise reduction and 12-bit signal resolution with a linear
output, should be employed ( 44 ). The linear output feature refers
to a direct linear relationship between the number of detected
photons and the corresponding pixel values. Compared to assess-
ing synaptic activation using IOS, SD is a slow process and low
frame rates will suffi ce (e.g., 5 Hz or less) to monitor the tempo-
rospatial feature of the spread of SD. A stabilized light source,
equipped with a xenon or halogen lamp, should be used to avoid
fl uctuations which would interfere with the analysis of the IOS. In
general, image acquisition is followed by processing of the image
data consisting of subtraction of the images which are then over-
layed (pseudocolored) on the original image (Fig. 2 ). Regions of
interest (ROIs) can be defi ned and the average light intensity
within the ROI is determined over a time series of images. In our
laboratory, optical changes are monitored by transilluminating the
slices from below using a halogen lamp and a stabilized voltage-
regulated power supply (KL 1500, Schott). Homogenous illumi-
nation is achieved by an arched glass rod built into the interface
chamber that conducts the light to the transparent fl oor of the
chamber. Light transmission changes are recorded using a micro-
scope-mounted camera (DFC 360FX, Leica) that yields 12-bit bit-
map image fi les which are acquired at a frame rate of 5 Hz, digitized,
and stored for further processing. The fi rst frame ( T 0 ) in a series,
captured before the onset of SD, serves as control and is subtracted
from subsequent images of the series ( T = T x − T 0 ) and divided by

the control image to obtain the relative light transmission change:

Δ

LT

L −

LT

Δ LT (%) =

i 0

× 100

i

0

ROIs are placed around the recording electrodes to quantify
LT changes. SD velocity is then determined by the propagation of
the transient LT change.