Stroke. 1999;30:1134-1141
(Stroke. 1999;30:1134-1141.)
© 1999 American Heart Association, Inc.
Early Delineation of Ischemic Tissue in Rat Brain Cryosections by High-Contrast Staining
Johannes Vogel, MD;
Christian Möbius, BSc
Wolfgang Kuschinsky, MD
From the Department of Physiology, University of Heidelberg (Germany).
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Abstract
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Background and PurposeAfter
short periods of ischemia,
commonly used staining methods yield
only moderate differences
in optical contrast between normal and
damaged brain tissue
when gray-scale images are used for
computer-assisted image
analysis. We describe a high-contrast
silver infarct staining
(SIS) method that allows an early delineation
of ischemic tissue
as soon as 2 hours after middle cerebral
artery occlusion (MCAO)
in rat brain cryosections.
MethodsRats were subjected to permanent MCAO for 2, 4, 6, and 48
hours. The optical densities were quantified in nonischemic
white and gray matter and in damaged tissue from gray-scale images of
serial sections with the use of a video camerabased image analyzing
system. SIS, hematoxylin-eosin, Nissl, and nitroblue tetrazolium
stainings were performed in cryosections, and
2,3,5-triphenyltetrazolium hydrochloride
(TTC) staining was performed in unfrozen vibratome sections. In
addition, the range of reduced cerebral blood flow (CBF) in areas
demarcated by SIS was determined in iodo[14C]antipyrine
autoradiograms of adjacent cryosections.
ResultsAt all times after MCAO, only SIS showed significantly
(P<0.01) lower optical densities in damaged than in
normal brain tissue for both white and gray matter. TTC staining was as
effective as SIS 6 and 48 hours after MCAO. The tightest correlation
between areas of reduced SIS and of reduced CBF was found at a mean
ischemic CBF of 22.3 mL/100 g per minute. This corresponds to a
CBF range of 0 to 44 mL/100 g per minute in areas of reduced SIS.
ConclusionsIn contrast to other staining methods, SIS allows a
reliable delineation of ischemic brain tissue (core plus
penumbra) from nonischemic white and gray matter of rat brain
cryosections as soon as 2 hours after MCAO.
Key Words: cerebral ischemia, focal histology staining tetrazolium salts rats
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Introduction
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Clinical studies have demonstrated the importance of an
early
therapeutic intervention after the onset of
stroke.
1 2 As a
consequence, experimental models of stroke
are used to demonstrate
the effects of an early therapeutic
intervention on the size
of the brain lesion. To this end, the
boundaries of the lesion
are traced in black and white images of
stained brain sections
that have been acquired with the use of a video
camerabased
image analyzing system. However, gray-scale images of
brain
sections stained by conventional histological
methods such as
hematoxylin-eosin (H&E)
3 4 5 6 7 or toluidine
blue
4 only
show a moderate contrast between normal and
damaged tissue,
especially after short periods of ischemia.
This contrast is
lower than that which exists between gray and white
matter in
the normal nonischemic brain. Therefore, the lesion
cannot be
distinguished reliably from normal white matter structures by
a
lower optical density (OD) in black and white images of such
stains.
This results in an erroneous determination of the ischemic
area
with the use of computer-assisted image analyzing
systems.
8 The same problem, based on a lack of difference
between the
OD of normal white matter and ischemic brain
tissue, is also
typical of nitroblue tetrazolium (NBT)
9 10 11 and
2,3,5-triphenyltetrazolium
hydrochloride
(TTC)
7 12 13 14 15 stains. These stains are also
commonly used
for the delineation of ischemic brain tissue.
NBT and TTC
stains are based on the functioning of mitochondrial
enzymes.
Therefore, the intensity of these stains is related
to the number of
intact mitochondria. The low density of mitochondria
in white matter
structures
16 17 results in a pale stain. This
makes it
impossible to discriminate between normal white matter
structures and
ischemic brain tissue in NBT as well as TTC stains.
This is
particularly true for the first hours after onset of
ischemia.
8 18 An additional disadvantage of TTC
staining arises from the
fact that only native, unfixed tissue can be
used. Compared
with native tissue, cryofixed tissue offers some
advantages
since the same cryofixed tissue sample can be used for
simultaneous
analysis by various techniques, such
as histochemistry, histology,
autoradiography, and
molecular biology. Therefore, the present
study aimed to develop an
easy and rapid staining method suited
for computer-assisted
discrimination between normal and ischemic
brain tissue
by the use of gray-scale images obtained from stained
cryosections.
Such staining should result in large differences
in OD between
nonischemic and ischemic brain tissue and minimal
differences
between normal gray and normal white matter. To compare
this
staining with commonly used staining methods, the OD in gray
and
white matter and in the lesioned tissue should be determined
in
gray-scale images of H&E-, Nissl-, NBT-, and TTC-stained
sections with
the use of a computer-based image analyzing system.
For the detection
of the degree of oligemia and ischemia, local
cerebral blood
flow (CBF) should be measured within the damaged
tissue as delineated
by the new staining method with the use
of quantitative
iodo[
14C]antipyrine
autoradiography.
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Materials and Methods
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Surgery
The experiments were performed in 24 adult male Sprague-Dawley
rats
in accordance with institutional guidelines. Twelve rats were
used
for histology and 12 for local CBF measurements. The animals
were
anesthetized by a gas mixture containing 1% to 1.5%
halothane,
70% N
2O, remainder
O
2. Body temperature was maintained at 37°C
to
37.6°C with a temperature-controlled heating pad. Blood
flow to the
right middle cerebral artery was blocked by an intraluminal
nylon
thread (diameter 0.15 mm), which was covered with an elastomeric
impression
material (Provil, Bayer) at the end over a
length of 10 mm,
as described by Nagasawa and
Kogure.
19 Before the intraluminal
suture was introduced,
12 rats were equipped with catheters
inside the right femoral vein and
artery to enable the infusion
of
iodo[
14C]antipyrine for the measurement of
local CBF. After
the incisions were closed, the anesthesia
was withdrawn, and
the animals were housed in cages with free access to
food and
water.
Histology (TTC, NBT, H&E, Nissl)
Twelve of the rats were killed 2, 4, 6, or 48 hours after middle
cerebral artery occlusion (MCAO). The brains were cut immediately in a
vibratome at +4°C into 500-µm coronal sections during superfusion
with a Ca2+-free solution (mmol/L: NaCl 119.5,
KCl 3, MgCl2 1, NaHCO3 24,
NaH2PO4 1, glucose 10) that
was bubbled with 5% CO2/95%
O2. Alternating sections were placed either into
glass vials containing 1% TTC dissolved in 0.9% saline for 5 minutes
or spread out on brass disks (20-mm diameter, 3-mm thickness). After
these brass disks were frozen to -20°C, two 20-µm and two 10-µm
sections were cut from the 500-µm-thick brain sections in a
cryomicrotome at the same temperature and transferred to
polylysine (Sigma) precoated slides. One of the 20-µm sections
obtained from each brass disk was stained for the activity of succinate
dehydrogenase with the use of NBT, as described by Riddle et
al.20 The other 20-µm section was stained by the newly
developed silver infarct staining (SIS) method, as described below. The
10-µm sections were stained by H&E or Nissl after they were treated
with a 4% formaldehyde solution for 5 minutes
In preliminary experiments, SIS was also applied at shorter delays of
MCAO between 0.5 and 1 hour. However, after such short periods of MCAO,
SIS does not provide sufficient contrast between the lesion and normal
brain tissue.
Silver Infarct Staining
The silver staining method is a modification of a neurofibril
staining introduced by Hortega.21 A silver staining method
was chosen since proteolysis of fibrillic macromolecules that are
stained by silver starts a few minutes after onset of
ischemia.22 23 For SIS, the slides were submerged
for 2 minutes into a silver impregnation solution (see below), which
was shaken vigorously. Then the slides were washed in distilled water 6
times for 1 minute before they were transferred to a vigorously shaken
developer solution for 3 minutes (see below). After the slides had been
washed in distilled water 3 times for 1 minute, they were air dried.
The composition and preparation procedures of the impregnation and
developer solution were as follows.
Impregnation Solution (90 mL)
Ten milliliters of a saturated lithium carbonate solution was
added to 5 mL of a 10% silver nitrate solution. The formed precipitate
was dissolved during continuous stirring by addition of a 25% ammonia
solution (
500 µL) drop by drop until the solution became clear.
Then 75 mL distilled water was added, and the solution was kept under
darkness until use. The addition of ammonia is the most critical point
of the total staining procedure. Fine remnants of the precipitate do
not disturb the reaction, whereas a surplus of ammonia results in a
failure of the staining.
Developer Solution (105 mL)
Twenty milliliters of a 37% formaldehyde solution was mixed
with 70 mL of distilled water. Then 0.3 g hydroquinone and
15 mL acetone were added, and the hydroquinone was dissolved by
gentle agitation. Next, 1.1 g trisodium citrate dihydrate was
dissolved within this solution. Thereafter, this solution was exposed
to room air until it became copper colored (30 to 60 minutes). All
solutions were prepared daily in carefully cleaned (65% nitric
acid/distilled water) glassware.
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Autoradiography
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Two, 4, 6, or 48 hours after onset of MCAO, animals were
placed
into a rat restrainer (Braintree Scientific) for the measurement
of
local CBF by the autoradiographic method of Sakurada et
al,
24 as described elsewhere.
25 In brief, 125
µCi/kg body
wt iodo[
14C]antipyrine (Biotrend)
was infused with an increasing
infusion rate for 1 minute. Parallel to
this, 12 to 16 timed
arterial blood samples were taken for
the determination of the
time course of the arterial
iodo[
14C]antipyrine concentration.
At the end
of the infusion period, the animals were decapitated,
and the brains
were removed as quickly as possible and frozen
in 2-methylbutane
chilled to -60°C. Then the brains were
embedded in M-1 embedding
matrix (Lipshaw) and cut into 20-µm
coronal sections at -20°C in a
cryomicrotome. After they
were dried on a heating plate at +60°C, the
sections were
exposed together with a [
14C]
standard set on a Kodak MinR1
x-ray film for 21 days. From the OD of
the autoradiograms, local
CBF was calculated with the
use of an image analyzing system
(MCID, Imaging Research Inc). In the
autoradiograms, the size
of the visible area of the
total oligemic and ischemic tissue
and the size of the areas in
which the CBF was <30, 20, and
10 mL/100 g per minute were determined.
Sections directly adjacent
to those used for
autoradiography were stained by SIS as described
above.
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Data Analysis
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In the first experimental group, the different staining methods
were
compared. The OD in 3600 to 4000 pixels of gray and white matter
as
well as in the lesioned tissue was measured in adjacent SIS-,
NBT-,
H&E-, and Nissl-stained cryosections and TTC-stained
vibratome sections
of 3 rat brains 2, 4, 6, and 48 hours after
MCAO, respectively, with
the use of the aforementioned image
analyzing system (charge-coupled
device [CCD] camera used: Sony
XL-77CE). The average OD measured in
gray matter, white matter,
and the lesioned tissue was compared with
the multiple Student's
t test and Bonferroni correction.
This analysis was performed
for each different staining method.
In the second experimental
group, the autoradiograms
were analyzed for areas of reduced
blood flow. To this end, 4
areas of reduced blood flow were
classified in each
autoradiogram: the area of the total oligemic
and
ischemic tissue that was visible by a lowered OD and the
areas
that had CBF values ranging from 0 to 10, 0 to 20, and
0 to 30 mL/100 g
per minute were determined. These areas were
related to the area of
infarcted tissue marked by SIS in adjacent
cryosections with the use of
linear regression analysis. The
correlation coefficients
obtained were tested for their difference
from zero. The areas of
damaged (SIS) and ischemic (autoradiography)
tissue
were determined by a blinded investigator. The level of
statistical
significance was set at
P<0.05.
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Results
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The results of the OD measurements obtained by the different
staining
methods are summarized in Figures 1

and 2

.
Figure 1

gives examples
of OD profiles determined by the
different methods 2 or 48 hours
after MCAO at the same level of
consecutive serial sections.
Exclusively in SIS sections, the contrast
between gray and white
matter is minimal in nonischemic tissue,
whereas large differences
exist in the OD between normal and
ischemic brain tissue at
all times after MCAO. In all other
stains, the optical contrast
between normal gray and normal white
matter is higher or equals
that which exists between gray matter and
ischemic tissue, except
for the TTC stain 6 and 48 hours after
MCAO. In the NBT, H&E,
and Nissl stains, the ODs of nonischemic
white matter were comparable
to or even lower than those of
ischemic tissue up to 6 hours.
The same was found in TTC stain
only 2 hours after MCAO. Therefore,
only SIS allows the delineation of
ischemic brain tissue in
gray as well as in white matter
structures for all times after
MCAO. TTC appears to be as effective as
SIS 6 and 48 hours after
onset of ischemia. The summary of
these findings obtained from
all different staining methods is given in
Figure 2

.

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Figure 1. Examples of OD profiles obtained from adjacent
brain sections subjected to different staining methods 2 (a through e)
and 48 (f through j) hours after MCAO. OD was measured along the white
line crossing each section. The white line crosses comparable locations
of gray and white matter in the nonischemic and the lesioned
tissue in each section. The use of 20-µm (SIS, NBT) and 10-µm (H&E,
Nissl) cryosections (SIS, NBT, H&E, Nissl) and unfrozen vibratome
sections (TTC) resulted in slight irregularities of the different
sections. Therefore, the white line does not cross completely identical
pixels in all sections. In contrast to all other staining methods, the
OD in white and gray matter is the same in SIS-stained sections (a, f).
In addition, the differences in OD between normal and damaged brain
tissue are highest in SIS sections. The borderline between normal and
ischemic tissue is clearly visible. OD values measured within
the ventricle lumen are shaded because they are not relevant for the
present findings. Width of the sections=13 mm.
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Figure 2. ODs of nonischemic white matter (white
bars) and the lesioned tissue (black bars) related to that of
nonischemic gray matter (gray bars). For better comparison, the
OD of gray matter was set to 100% for all staining methods at all
times after MCAO. Exclusively in SIS sections, the lesioned tissue can
be distinguished from both gray and white matter at all times after
MCAO. Only SIS-stained sections showed the same OD in gray and white
matter, whereas in all other stains white matter had a lower OD. NBT,
H&E, and Nissl showed higher or equal ODs in the lesion than in
nonischemic white matter up to 6 hours after MCAO; this was
true for TTC only 2 hours after MCAO. With the exception of SIS-stained
sections, significantly lower ODs between the lesion and white matter
were only found in TTC-stained sections 6 and 48 hours after MCAO.
*P<0.05, **P<0.01.
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The Table
shows the
physiological variables of the rats subjected
to local CBF measurement at different times after MCAO. All measured
parameters remained unchanged. To assess the CBF in the
areas of reduced SIS staining, the areas of reduced blood flow were
related to the areas of reduced SIS intensity. Figure 3
shows typical
iodo[14C]antipyrine
autoradiograms of cryosections together with the
adjacent silver-stained cryosections taken 2 and 48 hours after MCAO.
The areas of low perfusion in the autoradiograms
correspond well with the areas of less intense staining in SIS. The
relationship between the areas of lowered OD in the
autoradiogram and SIS was quantified by linear
regression analysis for different times and degrees of
ischemia. Figure 4
shows 2
extreme examples of the correlations between the ischemic areas
determined in numerous pairs of silver-stained sections and
autoradiograms. One regression line refers to an early
measurement 2 hours after MCAO. From the
autoradiograms, areas have been selected in which CBF
was <10 mL/100 g per minute. The correlation coefficient
(r) of 0.62, although significant, indicates a considerable
scatter of the data points around the regression line. In addition, the
ischemic areas determined by SIS were approximately twice as
large as those that had a CBF of <10 mL/100 g per minute. In contrast,
the regression line obtained 48 hours after MCAO indicates a high
degree of congruence between the areas of low blood flow and SIS. For
this correlation, the total oligemic and ischemic areas have
been taken (r=0.97). The 16 correlation coefficients
(r) obtained for the different times after MCAO (2, 4, 6, 48
hours) and the corresponding values of CBF (total oligemic and
ischemic tissue, CBF <30, <20, <10 mL/100 g per minute) are
summarized in Figure 5a
. Obviously, the
correlations were weaker at low ischemic CBF values and short
times after MCAO. With increasing duration of MCAO, the correlation
between the area of infarcted tissue determined in SIS sections and the
areas of ischemic CBF determined by
autoradiography also became tighter (higher
r) for lower mean ischemic CBF values.

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Figure 3. Comparison of SIS (a, c) and
[14C]iodoantipyrine autoradiography (b,
d) 2 (a, b) and 48 (c, d) hours after MCAO. The areas of damaged tissue
visible by reduced SIS (a, c) correspond well with the areas of low
perfusion in the autoradiograms (b, d). In contrast to
autoradiography, SIS (a, c) staining of
nonischemic white matter is as intense as in
nonischemic gray matter. This allows the exact delineation of
ischemic tissue by SIS in white matter structures as well. For
example, note the small tip of nonischemic white matter in the
corpus callosum 48 hours after MCAO (c, arrowhead), which cannot be
recognized in the corresponding autoradiogram (d).
Bar=5 mm.
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Figure 5. Correlation coefficients (a) and slopes (b) of the
regression lines that describe the relationships between the areas of
damaged tissue determined by SIS and by autoradiography
(z axis) at different values of mean ischemic
CBF (6.3, 11.2, 15.8, and 22.3 mL/100 g per minute; y
axis) after different periods of MCAO (2, 4, 6, and 48 hours;
x axis). a, Correlation coefficients of the regression
lines. All correlation coefficients were significantly different from
zero (P<0.01). The correlations between SIS and blood
flow became tighter with higher ischemic blood flows as well as
with longer periods of MCAO. b: Slopes of the regression lines. Slope
values closest to 1 were found at a mean ischemic CBF of 22.3
mL/100 g per minute, indicating that the areas of damaged brain tissue
determined by SIS are nearly congruent with the areas of low blood flow
determined by autoradiography. At lower blood flows,
the lower slopes indicate an overestimation of the ischemic
areas by SIS.
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The correlation coefficient r allows one to quantify the
scatter around the regression line, but it does not contain information
about the range of reduced blood flow that corresponds to an area of
infarcted tissue determined by SIS. This relationship can be quantified
by the slope of the regression lines. A slope of 1 indicates a
congruence of the areas measured in SIS sections and the corresponding
areas of ischemic CBF range. Figure 5b
shows the slope
values of the 16 regression lines obtained for 4 ranges of blood flow
and 4 periods of ischemia. Regression lines were closest to the
line of identity (0.98, 0.91, 1, 1.14) for areas that covered the total
oligemic and ischemic tissue (mean ischemic CBF of 22.3
mL/100 g per minute). This shows that SIS delineates the total oligemic
and ischemic tissue as soon as 2 hours after MCAO and
later.
In addition to the mean blood flow that exists in the areas detected by
reduced SIS, it is of interest to estimate the range of flow values
within these areas. To this end, the mean values of reduced CBF were
related to the ranges of reduced CBF as measured in the corresponding
areas (Figure 6
). This relationship was
based on the fact that the areas of the total oligemic and
ischemic tissue visible in the autoradiograms
are nearly congruent with the areas of lowered staining by SIS (Figure 5b
). These visible areas of the total oligemic and
ischemic tissue in the autoradiograms had a
mean CBF of 22.3 mL/100 g per minute. When areas of lower flow ranges
were selected (0 to 10, 0 to 20, and 0 to 30 mL/100 g per minute),
these areas had corresponding lower values of mean CBF (6.3, 11.2, and
15.8 mL/100 g per minute). These ranges of ischemic CBF were
then correlated with the corresponding mean ischemic CBF values
with the use of linear regression analysis. The resulting
regression line was used to estimate the range of blood flows that can
be expected in the area of diminished SIS. Regression analysis
showed that the mean ischemic CBF of 22.3 mL/100 g per minute
as detected from the reduced density in the
autoradiograms corresponds to a range of blood flows
from 0 to 44 mL/100 g per minute (Figure 6
). Such an
extrapolation was necessary since the direct measurement of areas
displaying blood flow values between 0 and 44 mL/100 g per minute in
the autoradiograms of ischemic tissue could be
misleading: these areas could include normal, nonischemic white
matter of corresponding blood flow values.25

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Figure 6. Relationship between the ranges of reduced CBF and
mean values of reduced CBF. This relationship was used to estimate the
range of flow values that is detected by a reduced intensity of SIS.
The basis of this estimation was the fact that at a mean CBF of 22.3
mL/100 g per minute, the areas of lowered staining by SIS were nearly
congruent with the areas of low blood flow determined by
autoradiography (Figure 3 ). A regression line was
calculated for the mean ischemic CBF values measured in the
areas in which CBF was <10, 20, or 30 mL/100 g per minute. The
equation of this regression line
(y=0.475x+1.6; r=1) can be
used to calculate that a mean ischemic CBF of 22.3 mL/100 g/min
corresponds to a range of blood flows from 0 to 44 mL/100 g per minute.
The direct measurement of areas displaying blood flow values >35 to 40
mL/100 g per minute in ischemic tissue is misleading because it
includes normal, nonischemic white matter25 (see
Discussion).
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Discussion
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According to the present data, gray-scale images of SIS appear
to
be superior to other staining methods for tracing infarct size,
especially
at early times after MCAO. In contrast to all other staining
methods
tested, SIS showed a large and well-detectable difference in
OD
between damaged and normal brain tissue and no differences
between
white and gray matter as soon as 2 hours after MCAO.
Comparison
of the size of SIS-negative areas in brain cryosections
with the
corresponding area sizes of reduced CBF in
iodo[
14C]antipyrine
autoradiograms
2 to 48 hours after MCAO showed a
significant correlation of
both area sizes for all times. The
correlation became tighter
at longer periods of MCAO and at higher
blood flow values in
the ischemic area.
In studies dealing with focal ischemia, lesion boundaries
between normal and ischemic tissue have frequently been
determined on the basis of the OD differences in black and white images
of stained brain sections that have been acquired with a CCD camera
connected to an image analyzing system.8 18 In such an
analysis of sections stained by commonly used staining methods,
the optical contrast between normal and ischemic tissue is low
for several reasons: (1) Cellular changes such as edema and
nuclear shrinking, when detected by conventional
histological methods (eg, H&E or Nissl), can only be
detected as moderate changes of the OD in gray-scale images. (2)
Histological changes during ischemia often
consist of a shift in the color distribution, which may not be
detectable as a change in OD since different colors, eg, red and blue
in H&E-stained sections, may result in similar gray values. This effect
can be increased because the sensitivity of CCD cameras is proportional
to the wavelength of the light at <600 to 700 nm. Therefore, the blue
stains (eg, Nissl or NBT) in particular show a poor signal-to-noise
ratio when acquired with a CCD camera. (3) The OD of
nonischemic white matter is low (Figures 1
and 2
). Since infarcted tissue is recognized by its reduced OD, it
is almost impossible to distinguish between white matter structures and
infarcted tissue with the use of image analyzing systems.8
As long as rat brain is used for studies of cerebral ischemia,
the error of the determination of the ischemic area arising
from white matter is small since rat brain contains
10% white
matter. For ischemic brains of other species such as cats or
primates, which have a higher percentage of white matter, this appears
to be a more serious problem.
In the present study the most commonly used staining methods were
compared with SIS. To the best of our knowledge, no staining method
exists that provides a significant optical contrast in gray-scale
images between normal and ischemic brain tissue (gray and white
matter) as soon as 2 hours after MCAO. In contrast to all other
staining methods tested, the SIS method presented here yields
the following advantages: (1) An equal stain of gray and white matter
is found in nonischemic tissue and, at a lower OD, in
ischemic tissue (Figures 1
and 2
). Although the
SIS method has not yet been tested in cats or primates, it appears
likely that the same results can be obtained from these species. (2) A
high optical contrast between normal and ischemic tissue is
found in SIS sections 2 hours after MCAO, whereas such a high contrast
is not obtained by NBT, H&E, and Nissl 48 hours after MCAO. (3) SIS is
based on black and white staining, which makes it independent of the
color sensitivity of CCD cameras. Thus, SIS allows one to distinguish
ischemic from normal brain tissue earlier and more exactly than
all other staining methods tested. Therefore, the lesion boundaries of
the damaged brain tissue can be detected automatically with a high
degree of accuracy that is not influenced by the subjectivity of the
observer.8 18
Additional advantages of SIS derive from the fact that SIS can be
performed in frozen tissue. Compared with TTC, which is frequently used
for the detection of ischemic brain tissue, this has 2
consequences: (1) A higher spatial resolution can be achieved by SIS
since thinner tissue slices (20-µm cryosections versus 0.5- to 2-mm
vibratome/razor blade sections) can be obtained. (2) Since other
methods of tissue processing are based on the use of cryosections or
can be performed in cryofixed material, the SIS method enables the
investigator to directly relate the infarct size to
parameters of tissue function, such as histochemical,
immunological, or perfusion-related data, which can be measured in
adjacent cryosections. The detection of multiple parameters
in adjacent brain sections within the same animal is especially useful
in models of focal brain ischemia because the extent of
infarction varies considerably from animal to
animal.26
Previously, thresholds of CBF have been defined to specify the
pathological changes that occur in rat brain tissue after MCAO.
Cerebral protein synthesis was found to be inhibited at CBF values <50
to 55 mL/100 g per minute 1 to 12 hours after MCAO.27 28
Tissue acidosis was detected at CBF values <30 mL/100 g per minute,
whereas energy depletion started to occur at <15 to 20 mL/100 g per
minute in rats subjected to 2 hours of MCAO.27 28 29
Therefore, it was of interest to define at which values of lowered CBF
SIS intensity was reduced. Qualitatively, SIS was visibly reduced in
all areas of clearly reduced CBF in the adjacent
autoradiograms. Quantitatively, areas that had CBF
values ranging from 0 to 10, 0 to 20, and 0 to 30 mL/100 g per minute
were smaller than the corresponding lesion size determined in SIS
sections, indicating that SIS includes CBF values >30 mL/100 g per
minute. Inclusion of areas of CBF values ranging from 30 to 40 mL/100 g
per minute would result in an erroneous inclusion of
nonischemic white matter structures by the image analyzing
program since nonischemic white matter has blood flows of
35
to 40 mL/100 g per minute.25 Therefore, lesion size was
determined in iodo[14C]antipyrine
autoradiograms by manual tracing of the visible areas
of the total oligemic and ischemic tissue (gray and white
matter). For all times after MCAO, these areas (mean ischemic
CBF, 22.3 mL/100 g per minute) closely correlated with the lesion areas
determined in corresponding SIS cryosections. However, the question of
which CBF range might correspond to a mean ischemic CBF of 22.3
mL/100 g per minute remained. Because of the potential inclusion of
nonischemic white matter, the CBF range in the tissue displayed
by SIS could not be determined directly. Therefore, the range of CBF in
the area displayed by SIS was estimated with an extrapolation from the
values of mean ischemic CBF of 6.3, 11.2, and 15.8 mL/100 g per
minute, which were derived from the areas that had CBF values ranging
from 0 to 10, 0 to 20, and 0 to 30 mL/100 g per minute. The mean
ischemic CBF values were related to their corresponding CBF
range (Figure 6
). The equation of the resulting regression line
was used to calculate the range of CBF values that corresponds to the
mean CBF of 22.3 mL/100 g per minute measured in the total oligemic and
ischemic tissue. This estimation yielded blood flow values
ranging from 0 to 44 mL/100 g per minute within the area of reduced SIS
intensity. Since the penumbra has been assigned to CBF values between
23 and 47 mL/100 g per minute,30 31 reduced SIS intensity
appears to encompass the total ischemic and oligemic (penumbra)
areas.
The present study shows that SIS is a sensitive staining method for
the detection of oligemic and ischemic brain tissue at CBF
values <44 mL/100 g per minute. In contrast to TTC, the new silver
staining method can be applied to cryosections and is suited for the
detection of oligemic and ischemic brain tissue as soon as 2
hours after onset of focal ischemia. This makes it possible to
combine SIS with autoradiographic, histochemical, and other
methods applied to adjacent cryosections of the same brain.
 |
Footnotes
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|---|
Reprint requests to Dr J. Vogel, Department of Physiology, University
of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg,
Germany.
Received September 21, 1998;
revision received January 25, 1999;
accepted January 27, 1999.
 |
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Editorial Comment
William I. Rosenblum, MD, Guest Editor
Department
of Neuropathology,
Virginia Commonwealth University,
Medical College of Virginia,
Richmond, Virginia
 |
Introduction
|
|---|
The authors describe a silver staining method, which, when coupled
with
the required image analyzing equipment, give an optical density
signal
that, by virtue of its pallor, distinguishes normal from
ischemic
rat brain. The method is used on frozen material, and in the
present
instance, employed frozen sections of the entire rat brain.
The
authors review other techniques for delineating ischemic
from
nonischemic brain. They report that the present technique
can
distinguish ischemic from nonischemic tissue at 2 hours,
a period
earlier than that reported for other techniques and
earlier than that
of a tetrazolium staining method or of Nissl-stained
or H&E-stained
sections used for comparison in the present
study. The new method, like
previous methods, is designed to
enable measurements of the size of
ischemic areas, presumably
so that one can compare effects of different
periods or severity
of ischemia or compare the effects of treatments on
the size
of the ischemic zone.
The new technique should be useful, especially for determining the size
of the "lesion" in the period 2 to 10 hours after injury. However,
the article raises interesting questions. First: what is being stained
by the precipitated silver? Silver stains, depending upon the recipe
used, can stain one or another type of glia, or they can stain neuronal
cell bodies or their axons and dendrites, or they can stain blood
vessels. The authors do not tell us what is being stained here, but it
may be the axons, since the white matter and cortex are both optically
dense until affected by ischemia, when both become pale and readily
distinguished from surrounding normal tissue.
Second, the authors express the belief that the technique will be
useful in animals with brains larger than those of rats. However, they
have not used other species. The tetrazolium techniques are often used
on gross (ie, not microscopic) slices of the brain. Using the entire
brain in that way, the investigator is not dependent on microscopic
sections and can delineate infarct volumes in large specimens. The
silver technique does not do this. Therefore, it would seem that its
value is, in fact, limited to brains sufficiently small so that an
entire hemisphere can fit on a slide after freezing and sectioning.
An important part of the study was the comparison of the staining
results with radioautographic determination of blood flow. The authors
discovered that pallor in the silver-stained sections corresponded to a
wide range of reduced flows, including those used by others to define
the penumbra. If this is correct, the authors have devised a method by
which the entire "at-risk" zone of ischemia can be morphologically
delineated at a period as early as 2 hours after ischemia. This could
then be compared with the volume of brain that actually dies with the
passage of time, and investigators could determine whether, and how
much of, the penumbra was saved. Two hours is an extremely early period
at which to define the area at risk. Indeed, microscopic evidence of
neuronal damage such as microvacuolation of neurons is difficult to
detect even in carefully perfused fixed brain prior to 4 hours, and the
much more readily recognized irreversibly injured eosinophilic or
acidophilic neuron may not appear until 12 to 24 hours after the onset
of ischemia, though some have reported seeing such neurons as early as
6 hours after ischemia. One wonders whether a careful comparison of the
pale area in the present study, with conventionally stained sections,
might reveal a heretofore unrecognized histological marker for cells
that are already irreversibly committed to die or (less likely, I
suppose) for cells that are still alive but malfunctioning in the
penumbra. In this regard, it is important to point out that the new
method, unlike some others, gives a strong change in optical density of
white matter shortly after onset of ischemia. Because neuronal cell
bodies are only present in gray matter, the signal in white matter must
reflect staining of one of the other elements mentioned above. If that
element is axons, and if the penumbra includes white matter (does it?),
then the present study shows that axons of neurons in the penumbra are
directly affected by ischemia and undergo some sort of reversible
alteration marked by failure of silver staining or that some change in
the neuronal cell bodies from which these axons arise has led to a
change in the argyrophilia of the axons. For this reason alone it would
be of great interest to pursue an understanding of the basis for the
staining and its failure in this study.
Received September 21, 1998;
revision received January 25, 1999;
accepted January 27, 1999.
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