lack of experience-mediated differences in the immunohistochemical expression of blood–brain...
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Developmental Brain Researc
Research report
Lack of experience-mediated differences in the immunohistochemical
expression of blood–brain barrier markers (EBA and GluT-1)
during the postnatal development of the rat visual cortex
Enrike G. ArgandonaT, Harkaitz Bengoetxea, Jose V. Lafuente
Laboratory of Clinical and Experimental Neuroscience (LaNCE), Department of Neuroscience, School of Medicine,
Euskal Herriko Unibertsitatea/University of the Basque Country, Leioako Campusa, Leioa E-48940, Spain
Accepted 15 February 2005
Available online 1 April 2005
Abstract
The development of the cortical vascular tree depends on functional development. External inputs are an essential requirement in the
modeling of the visual cortex, mainly during the critical period, when congruous blood supply is needed. The blood–brain barrier (BBB)
function regulates the passage of substances between the blood and the brain parenchyma, which is one of the main differential features of
central nervous system (CNS) microvessels. The endothelial barrier antigen (EBA) has been reported as a specific marker for the BBB
physiological function in rats. We studied the postnatal development of EBA expression in the visual cortex of rats reared under opposite
paradigms of visual experience, e.g., standard laboratory conditions, dark rearing, and enriched environment at 14, 21, 28, 35, 42, 49, 56, and
63 days postnatal (dpn). Parallel sections were immunohistochemically processed for endothelial barrier antigen (EBA) and glucose
transporter-1 (GluT-1). Total vasculature was quantified by Lycopersicon esculentum (LEA) lectin histochemistry. No differences in EBA
expression were found between groups, although quantitative differences were recorded paralleling differences in vascular density.
Paradoxically, there was no expression in certain cortical vessels which were GluT-1 immunopositive and positivity was consistent in non-
barrier areas such as the pineal gland. These findings were completely independent of age or experimental conditions. Therefore, the role of
the EBA antigen in the BBB remains unclear: it has been undeniably linked to vascular permeability, but its presence in non-barrier vessels
suggests another vascular function. Although visual experience modifies vascular density in the visual cortex, it has not been shown to have
an influence on the maturation of the BBB function.
D 2005 Elsevier B.V. All rights reserved.
Theme: Cellular and molecular biology
Topic: Blood–brain barrier
Keywords: BBB; Endothelial cell; Brain microvessels; Cerebral angioarchitecture; Pineal gland
1. Introduction
Postnatal development of the visual cortex is modulated
by experience. Extrinsic cues act as epigenetic factors in
concert with intrinsic developmental programs to shape
functional and structural cortical architecture [23]. Experi-
ence-mediated changes produce an increase in neuronal
0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.devbrainres.2005.02.007
T Corresponding author. Fax: +34 94 4649511.
E-mail address: [email protected] (E.G. Argandona).
URL: http://www.ehu.es/LaNCE/.
activity, which leads to increased metabolic demands [8,38]
involving the establishment of adaptive changes to meet new
requirements, such as changes in the vascular network [6,9].
Most of the changes induced by experience occur during a
defined time-window of postnatal life, called the critical
period; in rats, this period is between the 3rd and 5th weeks
postnatal, with the peak of maximum experience-induced
changes occurring between the 4th and 5th weeks [13].
The vasculature of the cerebral cortex is mainly
comprised of a capillary network which spreads throughout
the cortex [6], with differential characteristics as compared
h 156 (2005) 158 – 166
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 159
to vasculature outside the brain, and even as compared to
some other brain regions such as the blood–brain barrier
(BBB). The BBB is the set of physical and metabolic
mechanisms regulating the passage of substances between
the blood fluid and the cortical parenchyma. Most of these
mechanisms are involved in the regulation of fluid perme-
ability and are based on endothelial cells.
Some of these enzymatic mechanisms have been used to
quantify vascular distribution, such as Butyryl cholinesterase
histochemistry [3], alkaline phosphatase histochemistry [14],
LEA lectin histochemistry [12,21,37], and immunohisto-
chemistry against antigens such as glucose transporter-1
(GluT-1) [18], PECAM [24], RECA-1 [26], and the
endothelial barrier antigen (EBA) [11,19,25].
The EBA has been described as a protein triplet of 23.5,
25, and 30 kDa located in the luminal membrane of
endothelial cells [35]. Although its function remains
unclear, some studies have related the transient disappear-
ance of its expression in several experimental models to
pathological conditions involving a permeability increase in
allergic encephalomyelitis [36], brain injury [18,20], and
toxic administration such as sarin [1,2] or Clostridium
perfringens toxin [39]. EBA expression is therefore restored
in tandem with the recovery of the barrier function after
those episodes. For this reason, the EBA has been proposed
as a valid marker to study the brain vascular network with
their BBB mature and physiologically fully functional.
The development of the cortical capillary network is
closely linked to the development of the cortex, as capillary
density parallels local function. In previous work, we have
reported the influence of visual inputs on the postnatal
development of the vasculature of the striate cortex, showing
a delay in the maturation of the microvascular pattern and a
decrease in vascular density in dark-reared rats [3,4].
However, some authors have reported that microvascular
development remains after the end of the critical period in rats
reared in an enriched environment, and have also quantified
an increase in vascular density in enriched environments
[30,34].
The present work compares the postnatal development of
EBA expression in rats deprived of visual inputs and rats
reared in an enriched environment to standard reared rats in
order to try to evaluate the influence of visual experience
during the critical period. These hypothetical differences in
EBA expression and/or distribution at a given age could
suggest different stages in the maturation of the BBB depend-
ing on experience. As it has been described, the immunopo-
sitivity of the EBA can be linked to a maturational stage when
the BBB acquires the adult function. This positivity also
appears after the pathological situations described above and
is a sign of the recovery of the physiological function.
The EBA labeling appears at the same time as another
BBB marker, s-laminin [11], at the end of the 2nd postnatal
week. Therefore, the development of the adult distribution
pattern is completely coincident with the beginning and the
end of the critical period for the visual cortex.
Quantification has been performed on cortical layer IV,
where experience-induced changes occur, as this layer is the
termination site of visual afferents coming from the
thalamus [28,33]. Some authors have previously found
important dark-rearing-induced synaptic [10,22,31,32],
astroglial [5], molecular [7], genetic [17], and vascular
[4,14] changes on this layer; therefore, experience-induced
changes in EBA expression signaling BBB changes should
be found on layer IV.
2. Materials and methods
2.1. Animals
Three series of pregnant Sprague–Dawley rats were
raised in one of three rearing conditions:
– Five pregnant rats were placed in a dark room at the
beginning of pregnancy. Litters were born in complete
darkness, weaned at 21 days postnatal (dpn), and eight rats
of both sexes were sacrificed at each of the following ages:
14 dpn, 21 dpn, 28 dpn, 35 dpn, 42 dpn, 49 dpn, 56 dpn, and
63 dpn.
– Five pregnant rats were placed in an enriched environ-
ment consisting of a large cage full of colorful toys and
complex objects that were changed every 2 days. Eight rats
of both sexes were sacrificed at each of the above-mentioned
ages.
– Eight rats of each age raised in standard conditions (12-h
light–dark cycle) were sampled as control.
Water and food were available ad libitum for the three
groups. Every effort was made to minimize animal suffering
and to reduce the number of animals used. All animal
experiments were performed in accordance with the Euro-
pean Community Council Directive of 24 November 1986
(86/609/EEC).
Animals were anaesthetized with 6% chloral hydrate
(performed under dim red light for the dark-reared group).
After anesthesia, the animals were transcardially perfused
with a fixative containing 4% paraformaldehyde in 0.1 M
phosphate buffer. Perfusion was carried out with a pump at a
constant pressure of 20 mm Hg. Following perfusion, brains
were stored overnight at 4 -C in fresh fixative. The following
day, a thick block of occipital cortex containing the visual
area was removed sagittally with a Rodent Brain Matrix
(Electron Microscopic Sciences, USA), rinsed in cold PBS
for 4 h, and embedded in paraffin. The block was serially cut
with a microtome into sections of 4 Am and mounted on
slides coated with APES (3-aminopropyltriethoxylane).
2.2. Histochemical procedures
Paraffin was removed from the tissue through xylene
immersion; the tissue was rehydrated with ethanol. Endo-
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166160
genous peroxidase activity was blocked by incubation in 4%
H2O2/methanol for 20 min. Sections were washed in Tris
buffer at pH 7.4 and incubated overnight with biotinylated
lectin from Lycopersicon esculentum (LEA 5 Ag/ml;
SIGMA L0651) at 4 -C. Sections were rinsed with a Tris
buffer and incubated with avidin biotin peroxidase complex
(Elite ABC kit, Vector laboratories, Burlingame, CA, USA).
Finally, the reaction product was detected using 3,3¶-
diaminobenzidine (DAB 0.25 mg/ml) and hydrogen per-
oxide solution (0.01%). Slides were lightly counterstained
with hematoxylin, dehydrated, and covered.
2.3. Immunohistochemical procedures
Paraffin was removed from the tissue through xylene
immersion; the tissue was rehydrated with ethanol. Endo-
genous peroxidase activity was blocked with 4% H2O2/
methanol for 20 min. Sections were kept in a Tris buffer at pH
7.4, then incubated for 30min at 37 -C in Trypsin 0.1%CaCl2for antigen retrieval. Sections were incubated overnight with
an anti-EBAmonoclonal antibody (SternbergerMonoclonals
SMI-71; working dilution: 1:2000) and anti GluT-1 poly-
clonal antibody (Chemicon AB 1340 working dilution:
1:1000). Biotinylated secondary antibodies were used (Vec-
tor, USA) and the immunohistochemical reaction was
revealed by the avidin-biotin complex using diaminobenzi-
dine as a chromogen. Sections were lightly counterstained
with hematoxylin and finally dehydrated and covered.
Some sections were used to co-localize EBA and GluT-1
antigens. These sections were incubated overnight with the
aforesaid primary antibodies and with the following secon-
dary antibodies: FITC conjugate anti-mouse IgG (Ref: F-
9137 Sigma; working dilution: 1:100) and TRITC conjugate
anti-rabbit IgG (Ref: F-0382 Sigma; working dilution:
1:100).
Also included in each staining run were negative controls
in which the primary antisera and the lectin were omitted.
Images were acquired for confocal fluorescence micro-
scopy with an Olympus Fluoview FV500 confocal micro-
scope using sequential acquisition to avoid overlapping of
fluorescent emission spectra.
2.4. Morphometric procedures
To quantify changes that occurred during development
and to compare the three situations (normal, enriched
environment EE, and dark rearing DR), a blind morphometric
study was performed. In this study, the individual who
measured the sections did not know the features of each case
(neither the age of the rats nor whether they belonged to DR,
EE, or control groups). All sections of each case were
incubated for immunohistochemistry and histochemistry, and
the LEA, the EBA and the GluT-1 positive density were
estimated counting the number of positive vascular profiles
per area of visual cortex in serial sections. This was selected
with the aid of the Paxinos andWatson atlas [27]. Layers were
differentiated using parallel sections stained with Nissl, and
with hematoxylin counterstaining as a reference.
To estimate the number of profiles per area, the number of
positive capillaries present in an area delimited by a grid fixed
in the eyepiece was counted at 20� optical magnification,
excluding those intersected by both the x and y axes. The grid
was a square 250 Am in length, and thus the total surface was
62.500 Am2. The grid was randomly placed between cortical
layers III and V, ensuring that layer IV was included.
Measurements of each slice of the cortex were taken in
both hemispheres for each of the 10 slices taken per animal
(i.e., 60 fields per animal) and the mean value per animal
was calculated. The average values per group (8 animals)
were compared at each age by statistical analysis (ANOVA)
on Statview IIi Abacus Concepts.
3. Results
3.1. Qualitative results
3.1.1. LEA expression
LEA histochemistry showed a consistent staining of the
vascular network, which was homogeneously present on all
cortical layers, from I to VI; however, vessels were more
densely packed on layer IV. Within the cerebral cortex, there
was no regional variability in labeling, showing strong
staining of all vessels. The same expression pattern and
distribution were present at all ages (Fig. 1).
3.1.2. EBA expression
EBA immunostaining was homogeneously present
throughout the cortex on blood vessels of all sizes, although
some capillaries remained unstained. No other structures but
vessels were positive, including the neuropil. The expression
pattern was comparable to that of the LEA, although age-
related quantitative differences were found at the first two
ages.
At 14 dpn, only a small proportion of microvessels were
positive. From 21 dpn onwards, positivity was present in
most brain microvessels, but some cortical capillaries re-
mained unstained (Fig. 1). All the EBA-unstained capillaries
were GluT-1 positive (Fig. 2). Apart from cortical expression,
positive vessels were found in regions without barrier
function, such as pial vessels and especially the pineal gland
(Fig. 1h). No differences in pineal EBA expression were
found between different ages or experimental conditions.
Pineal positivity was as consistent as cortical positivity.
3.1.3. GluT-1 expression
All cortical blood vessels were immunopositive for GluT-
1, with an expression pattern similar to that of the LEA and
the EBA. Nevertheless, immunostaining was positive at all
the ages studied, with no qualitative differences related to
age or experimental condition. By contrast, no positive
vessels were found in the pineal gland (Fig. 1g).
Fig. 1. (a–h) EBA, GluT-1, and LEA histochemical and immunohistochemical detection of vessels in the rat visual cortex at several ages of postnatal
development. Arrows show EBA-negative and LEA-positive vessels. (a) EBA immunohistochemistry and (b) LEA histochemistry at 14 dpn. (c) EBA and (d)
LEA positivity at 28 dpn. (e and f) EBA and LEA at 63 dpn. (g) GluT-1 and (h) EBA immunohistochemistry in the pineal gland at 56 dpn. All images
correspond to control animals, although they were qualitatively indistinguishable from other groups. Scale bar is 50 Am in all cases.
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 161
3.2. Quantitative results
3.2.1. LEA
Vascular density underwent slight increases over the
course of postnatal development in the three experimental
groups, but quantitative differences were found from the
beginning of the critical period (Table 1 and Fig. 3b).
In dark-reared rats, vascular density was significantly
lower starting at the 5th week postnatal. Although at 14 dpn
there were 20% fewer vessels per area in dark-reared rats (23
vs. 29), differences disappeared at 21 dpn, when vascular
density was 8% lower in controls, although the difference
lacked statistical significance. At 28 dpn, density was (not
significantly) 5% lower in dark-reared rats; from 35 dpn
Fig. 2. (a–c) EBA and GluT-1 double labeling. Confocal fluorescent
images in c show co-localization (yellow) of both blood–brain barrier
markers in the visual cortex section of a 28-dpn control rat. (a) GluT-1
expression (red), (b) EBA expression showing the lack of staining in some
GluT-1-positive capillaries. (c) GluT-1 and EBA co-localization. Scale bar
is 50 Am.
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166162
onwards, differences were always significantly lower in dark-
reared rats: 9% at 35 dpn, 10% at 42 dpn, 8% at 49 dpn, 12%
at 56 dpn, and 7% at 63 dpn. In both conditions, the obtained
numbers were similar at all ages, ranging from 29 at 14 dpn to
36 at 63 and 49 dpn in controls, and from 23 at 14 dpn to 34 at
21 dpn in dark-reared rats.
Rats reared in an enriched visual environment showed
similar but opposite behavior, as vascular density was
higher than in controls. Greater differences were recorded
and also appeared earlier in the critical period, at the 4th
postnatal week.
At 14 dpn, environmentally-enriched rats showed 25%
fewer vessels per area than controls, a significant differ-
ence (22 vs. 29). As in the case of dark-reared rats,
numbers were similar at 21 dpn (mean value of 31.1 vs.
31.5), and from 28 dpn onwards, measurements were
always higher for the complex experience group: 29%
higher at 28 dpn; 11% higher at 35 dpn; 23% higher at 42
dpn; 19% higher at 49 dpn; 25% higher at 56 dpn; and
11% higher at 63 dpn. Although the numbers were again
similar for different ages within the same experimental
group, there was a major increase between 14 and 21 dpn
(21.6–31.1) and a lesser increase between 21 and 28 dpn
(31.1–37.7). Later, countings ranged from 37.7 at 28 dpn
to 42.4 at 49 dpn.
3.2.2. EBA
EBA cortical expression increased dramatically from 14
to 28 dpn, when it reached its adult level (Fig. 3a). Starting
at 21 dpn, significant quantitative differences were recorded
parallel to differences in vascular density.
Only 3.5 vessels per area were counted in controls at
14 dpn. However, there were already 23.1 by 21 dpn and
28.4 at 28 dpn. As of this age, EBA-positive numbers
remained close to those of the LEA (33 at 35 dpn; 32 at
42 dpn; 34 at 49 dpn; 31 at 56 dpn; and 35 at 63 dpn).
For rats reared in darkness, behavior was comparable to
controls, although with lower absolute values following the
measurements obtained with LEA. We recorded 29% fewer
vessels per area at 14 dpn, 22% more at 21 dpn (both values
were non-significant), and 11% lower vascular density at 28
dpn in dark-reared rats. This was 8.5% lower at 35 dpn;
13.6% lower at 42; 8% lower at 49 and 56 dpn; and 7%
lower at 63 dpn. Differences were significant from 28 dpn
onwards.
While EBA-positive vascular density was significantly
lower in animals reared in darkness from 28 dpn, it was
significantly higher in enriched experience-reared animals.
These results also corresponded to quantifications in total
vascular density measured with LEA histochemistry.
Numbers were similar to controls at 14 dpn in environ-
mentally-enriched rats, and they were 9% lower at 21 dpn.
From 28 dpn onwards, they were higher in controls: 23% at
28 dpn; 3.6% at 35 dpn; 12% at 42 dpn; 10% at 49 dpn;
28% at 56 dpn; and 10% at 63 dpn.
The percentage of negative vessels reached its max-
imum at 14 dpn when 88% of vessels remained unstained
in controls (89% in dark-reared rats and 84% in environ-
mentally-enriched rats) (Table 2). At 21 dpn, the percent-
age of positive vessels rose, and we recorded only 27% of
negative vessels in controls (17% in dark-reared rats and
32% in environmentally-enriched rats). As of 28 dpn, most
vessels were positive and the percentage of negatives was
less than 10% for all ages and experimental groups. At 28
dpn, negatives accounted for 3% in controls (C), 9% in
dark-reared rats (DR), and 7% in enriched environment
group (EE). At 35 dpn, it was 5% in C, 4% in DR, and
11% in EE. At 42 dpn, it was 2% in C, 6% in DR, and
11% in EE. At 49 dpn, it was 1% in C, 0.3% in DR, and
9% in EE. At 56 dpn, results showed 4% of unstained
vessels in C, 0.3% in DR, and 1.5% in EE. At the last
measured age, 63 dpn, it was 2.5% in C, 3% in DR, and
4% in EE (Fig. 3c).
Table 1
Positive vessel density along postnatal development (mean T SD)
Age EE DR Control DR vs. C (%) EE vs. C (%) p DR vs. C p EE vs. C
LEA
14 dpn 21.6 T 5.6 22.9 T 5.9 28.8 T 6.4 �20.5 �25 0.0001 0.0001
21 dpn 31.1 T 5.9 34.2 T 8 31.5 T 5.7 8.6 �1.3 0.06 0.75
28 dpn 37.7 T 6.7 27.9 T 6.6 29.2 T 3.7 �4.5 29.1 0.3 0.0001
35 dpn 38.3 T 5.3 31.5 T 6.7 34.6 T 5.9 �9 10.7 0.01 0.01
42 dpn 40.6 T 5.2 29.9 T 4.7 33.1 T 6.2 �9.7 22.7 0.004 0.0001
49 dpn 42.4 T 5.5 32.6 T 5.3 35.6 T 6.5 �8.4 19.1 0.01 0.0001
56 dpn 40.6 T 5.6 28.8 T 4.5 32.6 T 5.6 �11.7 24.5 0.0006 0.0001
63 dpn 39.8 T 9.2 33.2 T 4.6 35.7 T 6.5 �7 11.5 0.008 0.001
EBA
14 dpn 3.5 T 3.4 2.5 T 2.3 3.5 T 2.5 �28.6 0 0.04 0.9
21 dpn 21 T 6.4 28.3 T 8.9 23.1 T 4.9 22.5 �9.1 0.0008 0.1
28 dpn 35 T 8.5 25.3 T 4.2 28.4 T 4.3 �10.9 23.2 0.04 0.007
35 dpn 34.2 T 4.9 30.2 T 5.3 33 T 5.6 �8.5 3.6 0.02 0.4
42 dpn 36.3 T 8.6 28 T 5.8 32.4 T 3.6 �13.6 12 0.007 0.08
49 dpn 38.7 T 8.3 32.5 T 5.4 34.7 T 5.5 �7.7 9.9 0.04 0.04
56 dpn 40 T 5.1 28.7 T 3.1 31.2 T 4.8 �8 28.2 0.05 0.0001
63 dpn 38.2 T 5.7 32.3 T 5.4 34.8 T 7.4 �6.9 10.1 0.1 0.06
GluT-1
14 dpn 21.9 T 4.1 23 T 5.7 27.9 T 3.2 �17.6 �21.5 0.0001 0.0001
21 dpn 31 T 6.8 33 T 4.6 31.2 T 3.7 5.8 �0.6 0.0366 0.7594
28 dpn 37.8 T 5.3 27.9 T 4.5 29.1 T 4.4 �4.1 29.9 0.1827 0.0001
35 dpn 38.5 T 4.4 31.2 T 4.3 34.7 T 3.8 �10.1 11 0.0001 0.0001
42 dpn 40.1 T 5.2 30.4 T 5.2 33.9 T 3.8 �10.3 18.3 0.0005 0.0001
49 dpn 42.6 T 3.8 32.8 T 5.7 35.5 T 4.9 �7.6 20 0.0129 0.0001
56 dpn 41.9 T 3.3 29.4 T 4.4 33 T 4.4 �10.9 21.2 0.0001 0.0001
63 dpn 39.6 T 3.7 33.1 T 4.8 35.9 T 4.2 �7.8 10.3 0.0055 0.0001
Number of EBA-, LEA-, and GluT-1-positive vessels at various ages of development (dpn: days postnatal) for enriched (EE), dark-reared (DR), and control
groups (mean number of EBA- and LEA-positive vessels per 62,500 Am2 of visual cortex T standard deviation and statistical significance, P value).
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 163
Despite the variability of differences among the ages and
experimental groups, these differences were not statistically
significant.
3.2.3. GluT-1
Numbers were almost identical to the LEA ones at all the
ages and conditions. They have been reproduced in Table 1.
Fig. 3. (a–c) Comparison of average measurements between environmentally-e
Horizontal axes show the age of the animals. Vertical axes show: (a) number of E
positive vessels per 62.500 Am2 of visual cortex, (c) percentage of EBA-negative
show EC control significance.
4. Discussion
The postnatal development of cortical vascularization
consists of the establishment of a dense capillary network
dependent on the local function. In standard conditions, it is
completed around the end of the 4th postnatal week [30]. On
other hand, in animals reared under different environmental
nriched, dark-reared, and control groups at each of the ages considered.
BA-positive vessels per 62.500 Am2 of visual cortex, (b) number of LEA-
vessels in 62.500 Am2 of visual cortex; + show DR control significance, *
Table 2
Percentage of EBA-unstained vessels
Age EE (%) DR (%) Control (%) p DR vs. C p EE vs. C
14 dpn 83.8 89.1 87.8 0.013 0.0001
21 dpn 32.5 17.3 26.7 0.38 0.3
28 dpn 7.2 9.3 2.7 0.22 0.23
35 dpn 10.7 4.1 4.6 0.5 0.32
42 dpn 10.6 6.4 2.1 0.35 0.08
49 dpn 8.7 0.3 1.1 0.78 0.14
56 dpn 1.5 0.3 4.3 0.37 0.08
63 dpn 4 2.7 2.5 0.9 0.02
Number of EBA-negative vessels for enriched (EE), dark-reared (DR), and
control groups per 62,500 Am2 of visual cortex and statistical significance,
P value.
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166164
conditions, vascular plasticity remains after this date to meet
increased or decreased requirements [3,4,34]. We found no
qualitative differences during postnatal development
(before, during, and after the critical period) nor among
different experimental conditions. The main increase in
EBA staining occurred from 14 to 21 dpn, which suggests
that the function of the EBA is not linked to capillary
plasticity related to experience, since its expression is
established before the critical period, when most changes
occur. In previous studies using enzymes belonging to the
BBB such as Butyryl cholinesterase, we have found that
BChE histochemistry stains the endothelial wall all over the
cortex [3], which indicates that the maturation of the BBB is
a complex process with different rhythms for each
component. In the same way, the GluT-1 immunohisto-
chemical expression is also present in all cortical vessels at
all stages of postnatal development.
The quantitative effects of both visual deprivation and
increase of visual experience have already been reported in
previous works [3,4,34]. Nevertheless, the study was
performed on parallel slices to correlate this with the
EBA quantifications, as there are great differences in the
thickness of the samples and in the way the tissue is
processed. Therefore, in order to avoid a technical bias, the
quantitative analysis has been performed on the same
material processed in exactly the same way. The marker
used for this purpose was the Lycopersicon esculentum
(LEA), which in the cerebral cortex is localized to all
microvascular profiles, at the luminal surface of the
plasmalemma of the endothelial cells [21,29]. As expected,
significant differences were obtained within the critical
period when, in line with the above-mentioned literature,
vascular density was higher in enriched environment
animals and lower in dark-reared animals.
However, one new result should be noted: the effects of
increased visual experience seem to have quantitative results
at an earlier age than the lack of visual experience,
appearing 1 week in advance. The increase in vascular
density also doubles the decrease in dark-reared animals.
This finding could suggest that enriched experience not only
has effects related to visual experience, but also that its
effects could also be more complex, affecting the visual
cortex in different ways including microvascular differ-
ences. The effects of the complex experience itself on the
development of the vascular pattern of the visual cortex
should be the subject of further study.
On the other hand, although quantitative results in EBA
expression were obtained among experimental groups, they
reflected only differences in vascular density due to the
influence of visual experience, as described before. This is
corroborated by the fact that no differences were found when
comparing the density of unstained vessels. As in the case of
entire vascular density, differences among experimental
groups were recorded only after the beginning of the critical
period.
Previous studies have posited the EBA as a specific
marker for the BBB in rats, linking its expression with the
functionality of the barrier [19,39], since a decrease in EBA
positivity has been reported in pathological conditions
where the administration of an anti-EBA antibody resulted
in increased permeability of brain microvessels [15,16].
These findings have led to the postulate that the EBA is a
useful tool for quantifying cortical microvascularization, as
opposed to commonly-used antigenic markers such as GluT-
1 and non-antigenic markers such as UEA and LEA lectin
[19]. Despite the studies that have been conducted, the
EBA’s role in brain vessels remains unclear.
Our results point to a wider range of speculation about
the biological significance of the EBA and even about the
nature of the BBB function itself, since a significant
percentage of cortical vessels lack EBA expression while
non-barrier endothelium shows EBA positivity. The first
finding could suggest a discontinuity of the function along
the brain microvascular tree, whereas the second could point
to barrier antigenic expression in brain vessels without
barrier function. This is linked to brain vessels, since we
found no positivity in vessels outside the brain (in the liver
or kidneys, for example). We also examined the expression
of another component of the BBB, the Glucose transporter
GluT-1, which has been proven to remain positive even
when pathological alterations of the barrier promote changes
in EBA expression [18]. As was expected, no GluT-1-
positive vessel was found in the pineal gland whereas all the
cortical vessels were positive and the quantitative results
were identical to the LEA results.
Both these facts call into question the promotion of the
EBA as a marker for quantitative microvascular studies.
However, they also open up a new horizon for qualitative
studies on the role of the EBA, since it has been linked with
certainty to the endothelial function but its relationship with
the barrier function remains unknown. Some authors have
suggested a link between EBA expression and the influence
of the astroglial ensheathment, which shows the role of the
astroglia in the induction and/or maintenance of the BBB
markers in the cortex [11]. Our previous results on the
distribution of S-100-positive astrocytes in the visual cortex
of rats reared under different visual conditions showed a
diminishing of the astroglial density, but with an absence of
E.G. Argandona et al. / Developmental Brain Research 156 (2005) 158–166 165
qualitative differences. Therefore, these results agree with
both findings, as the influence of experience results in
quantitative rather than qualitative changes.
Therefore, further studies are required to correlate EBA
expression and elucidate its endothelial function.
Acknowledgments
This work was supported by the Basque Country
University Research Project UPV 078.352-EA 8042 and 9/
212.327-15887 Mr. H. Bengoetxea is supported by a Basque
Country University grant. Confocal microscopy analysis was
performed at the ‘‘Servicio de Microscopıa Analıtica y de
Alta Resolucion en Biomedicina’’ (High Resolution Ana-
lytical Microscopy Service for Biomedicine) at the Univer-
sity of the Basque Country.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.devbrainres.
2005.02.007.
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