Spreading Depolarization - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 331

image deconvolution software before obtaining dendritic spine
measurements. We apply a median fi lter (radius = 2) to reduce pho-
ton and PMT noise in images selected for presentations.
To assess changes in spines, individual sections through a
z-series of the dendrite are examined to ensure that they had not
rotated out of view or had become obscured by their dendritic
shaft or by one another. From individual sections of the image
stack, the maximal width of the spine head can be determined. The
brightness of the spine (measured as GFP fl uorescence) is propor-
tional to its volume ( 53 ), and relative volume could be estimated
as the spine fl uorescence divided by the dendritic fl uorescence
( 54 ). To correct for potential general changes in fl uorescence
intensity during SD caused by, for example, inconsistencies in exci-
tation, the average pixel value at the brightest point of spine image
is measured, background fl uorescence is subtracted, and the data
are normalized to the average peak fl uorescence of the largest den-
drite in the imaged fi eld.
Given the relatively poor axial resolution of 2PLSM (~2 m),

we use 2D maximum intensity projections (MIPs) of image stacks
for analyses of volume changes of neuronal and astroglial somata
during SD. Such analyses assume that soma volume is changing
uniformly in all directions. In this case, measurements of changes
in the lateral dimensions are adequate to determine relative volume
changes, which underestimate actual volume changes assuming
that they are approximately isotropic. We employ three techniques
to measure relative volume changes. (1) MIP images are digitally
traced by hand to measure the area of astroglial and neuronal soma
profi les in control and for each time point. (2) Control and experi-
mental MIP images are pseudocolored green and red, aligned, and
overlaid. Overlapping areas are projected as yellow, whereas differ-
ent areas remain red or green. (3) Control profi les are traced and
fi lled to create a mask, which reveals peripheral areas of swelling
when overlaid upon experimental images.
Dendritic beading with spine loss is considered an extreme ver-
sion of swelling by dendrites and when energy stores are severely
compromised, permanent dendritic beading with spine loss is a
sign of acute terminal injury ( 50, 51, 55-57 ). Dendritic beading is
identifi ed as the appearance of rounded regions extending beyond
the diameter of the parent dendrite separated by “interbead” seg-
ments. A spine is considered lost when there is no indication of a
spine head extending more than ½ of its diameter beyond the
beaded portion of the dendrite.
High-resolution time-lapse imaging during SD in metaboli-
cally compromised slices, as for example during oxygen-glucose
deprivation (OGD), presents some technical challenges since the
volume of the entire slice is dramatically altered, resulting in the
shifting of the focal plane. We, therefore, sample single-optical sec-
tions on the fl y while recentering and adjusting the fi eld of focus

μ