(Stroke. 1999;30:1134-1141.)
© 1999 American Heart Association, Inc.
Original Contributions |
Early Delineation of Ischemic Tissue in Rat Brain Cryosections by High-Contrast Staining
From the Department of Physiology, University of Heidelberg (Germany).
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Abstract |
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Background and Purpose—After 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.
Methods—Rats 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 camera–based 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.
Results—At 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.
Conclusions—In 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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TopAbstractIntroductionMaterials and MethodsAutoradiographyData AnalysisResultsDiscussionReferencesIntroduction |
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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 camera–based 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 blue4 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 structures16 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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TopAbstractIntroductionMaterials and MethodsAutoradiographyData AnalysisResultsDiscussionReferencesIntroduction |
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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% N2O, remainder O2. 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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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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y=0.5x-4.67), a large scatter of the
data points around the regression line was found at the low
ischemic CBF of <10 mL/100 g per minute. SIS yielded areas of
brain damage approximately twice as large as those detected by
autoradiography. In contrast, 48 hours after MCAO (
;
y=0.98x+0.006), the data points are close
to the line of identity (r and slope close to 1) when
SIS-stained areas were correlated to the areas of the total oligemic
and ischemic tissue (mean ischemic CBF, 22.3 mL/100 g
per minute), indicating that both methods yielded highly corresponding
areas of damaged brain tissue.
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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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Discussion |
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TopAbstractIntroductionMaterials and MethodsAutoradiographyData AnalysisResultsDiscussionReferencesIntroduction |
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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.
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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
Department of Neuropathology, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia
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TopAbstractIntroductionMaterials and MethodsAutoradiographyData AnalysisResultsDiscussionReferencesIntroduction |
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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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