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changes in the perihematomal brain, were investigated in relation
to different time points after induction of the hematoma. Depending
on the two study designs (1) sacrifi ce of the animal at defi ned time
points and postmortem brain investigation and (2) survival of the
animal and in vivo brain investigation, the tools to assess hema-
toma and edema size vary considerably. In this chapter, methods to
assess hematoma and edema volumes are reviewed.
2. Postmortem
Investigations
Morphometric analysis is a well-established method by which the
hematoma volume is calculated. For this analysis, whole brains are
harvested and then cut into thin slices. The slice thickness varies
substantially in the literature and also depends on the animals used
as well as the volume of the induced hematoma. The next step is to
obtain high-resolution images of each slice containing hematoma,
and then to calculate the cross-sectional areas of the clot on each
slice. Digital cameras and, in small animals, microscopes for mag-
nifi cation are used for that imaging. The hematoma volume is then
calculated by multiplying the cross-sectional areas with the dis-
tances between the sections and the number of the slices. Nowadays
this step is aided by software solutions. Several methods for prepar-
ing the brain for cutting into slices are used: freezing the brain in
liquid nitrogen immediately after removal, fi xating the whole brain
with a formaldehyde-containing solution prior to freezing or prior
to embedding into paraffi n.
Since the cross-sectional areas of the hematoma are either
obtained by manual segmentation or computer aided (
6
), a clearly
visible border zone between clot and normal brain is important.
Often the border zone with the reddish-brown clot and the gray-
ish-white brain parenchyma is suffi ciently identifi able for the mor-
phometric analysis. However, staining with 3,3ยข-diaminobenzidine
tetrahydrochloride (DAB) containing a metal enhancer, which
reacts with hemoglobin to form a blue-black product is used by
some to enhance the contrast (
7
). Methods that provide better
morphological information are Luxol fast blue counterstained by
cresyl violet (
8
), cresyl violet alone (
9
), and Nissl stain (
10
). With
Luxol fast blue, erythrocytes are stained blue, contrasting well with
cresyl violet which does not stain erythrocytes. With Toluidin blue
(modifi ed Nissl stain), erythrocytes appear dark blue whereas the
brain tissue is stained moderately. Hematoxylin and eosin stainings
reveal detailed morphological information, i.e., of the surrounding
tissue in an appropriate magnifi cation but do not show a contrast
between hemorrhage and brain tissue at lower magnifi cations. The
dehydration which occurs during the paraffi n embedding process
hardens the hemorrhages more than the brain tissue. This fre-
quently causes artifacts when pieces of hemorrhages break off
2.1. Evaluation
of Hematoma Volume
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