Microvascular Research 81 (2011) 153–159
Contents lists available at ScienceDirect
Microvascular Research
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Dynamics of dystroglycan complex proteins and laminin changes due to angiogenesis
in rat cerebral hypoperfusion
Edina A. Wappler a,b,⁎, István Adorján c, Anikó Gál a,d, Péter Galgóczy c, Kinga Bindics c, Zoltán Nagy a
a
Department Section of Vascular Neurology, Heart Center, Semmelweis University, Budapest, Hungary
Department of Anesthesiology and Intensive Therapy, Semmelweis University, Budapest, Hungary
Institute of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary
d
Clinical and Research Center for Molecular Neurology, Semmelweis University, Budapest, Hungary
b
c
a r t i c l e
i n f o
Article history:
Accepted 16 December 2010
Available online 28 December 2010
Keywords:
Cerebral hypoperfusion
Dystroglycan complex
Laminin
Angiogenesis
a b s t r a c t
Permanent bilateral carotid occlusion is a well known cerebral hypoperfusion model in rats. The aim of our
study was to investigate the different stages of vascular reaction by detecting changes in the extracellular
martix proteins and to examine their relationship to angiogenesis after occlusion. Experiments were
performed on adult male rats. Brain samples were investigated from day 1 to day 30 post-surgery.
Immunohistochemical analysis was performed on the whole hippocampus and on the adjacent cortex in
order to investigate extracellular martix proteins, such as the markers of dystroglycan complex (βdystroglycan, α-dystrobrevin and utrophin) and a marker of basal lamina (laminin). The levels of the proteins
were estimated by western blot analysis. Vascular density as well as blood–brain barrier permeability were
studied on brain slices from the same regions. Our results showed altered laminin and β-dystroglycan
immunoreactivity beginning 2 days after the onset of occlusion followed by an increased utrophin
immunoreactivity without blood–brain barrier disruption 5 days later. By day 30 of hypoperfusion, when
increased vascular density was detected, all changes returned to baseline levels. Western blot analysis showed
significant differences in β-dystroglycan and utrophin expression. Our results indicate that the different
stages of neovascularisation resulting from cerebral hypoperfusion can be well defined by the markers
laminin, β-dystroglycan, and utrophin and that these markers are more likely to correlate with glio-vascular
decoupling than does altered blood–brain barrier function.
© 2010 Elsevier Inc. All rights reserved.
Introduction
Permanent occlusion of both common carotid arteries in the rat is a
widely used model of cerebral hypoperfusion (for review, see e.g.,
Farkas et al., 2007). Cerebral circulation is re-established about 3
months following the occlusion of both internal carotid arteries,
however, in the acute phase (2-3 days after ligation), and in the
chronic/oligemic phase (8 weeks to 3 months after ligation), lower
cerebral blood flow was registered (Farkas et al., 2007). The complex
mechanisms that initiate and regulate the compensatory processes
are still unknown; however, it is probable that angiogenesis and
vascular remodeling together with changing vessel diameters are
crucial to the survival of brain tissue (Oldendorf 1989; Busch et al.,
2003; de la Torre et al., 2003; Choy et al., 2006; Paravicini et al., 2006;
Ohtaki et al., 2006; Farkas et al., 2007; Mracsko et al., 2010).
⁎ Corresponding author. Present address: Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Medical Center Blvd, Winston-Salem,
NC 27157, USA. Fax: + 1 336 716 0504.
E-mail addresses: [email protected], [email protected]
(E.A. Wappler).
0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.mvr.2010.12.005
Brain injuries provoke angiogenesis and/or arteriogenesis depending on the initial triggers. While hypoxia (through hypoxia-inducible
factor and vascular endothelial growth factor [VEGF]) triggers
angiogenesis, increased shear stress leads to arteriogenesis in the
brain tissue (Heil and Schaper, 2004; Heil et al., 2006; Schierling et al.,
2009). Permanent ligation of both carotid arteries induces changes in
pressure gradients across interconnecting arterioles, increases shear
stress and simultaneously induces hypoxia (Schierling et al., 2009).
Both mechanisms seem to be important in vascular remodeling in
this model. Following bilateral carotid occlusion, increased posterior cerebral artery and basilaris artery diameters were detected
(Schierling et al., 2009; Choy et al., 2006) together with a transient
proliferation of endothelial and adventitial cells in the same arteries
(with a peak on the 3rd day of the occlusion that decreased
dramatically by the 7th day of the occlusion) (Schierling et al.,
2009), as well as an increased number of vertebral collaterals (Choy
et al., 2006), with augmented cortical capillary diameter (Ohtaki et al.,
2006). In a three-vessel occlusion model (unilateral common carotid
artery occlusion combined with bilateral vertebral artery occlusion),
an increased posterior artery diameter was measured also at the side
of the carotid ligature (Busch et al., 2003).
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E.A. Wappler et al. / Microvascular Research 81 (2011) 153–159
During angiogenesis in the brain, glial cells and extracellular
matrix proteins such as laminin, tenascin-c, and fibronectin are
known to be relevant in the reorganization of the vessels (Lee et al.,
2009; Ohtaki et al., 2006; Tate and Aghi, 2009); however, the role of
dystroglycan complex (DGC) proteins is still not clear. DGC proteins
are known to be highly expressed on the foot processes of
perivascular astrocytes and are important in anchoring them to the
lamina basalis through laminin (Culligan and Ohlenieck, 2002). In
addition to the extracellular matrix binding of the DGC through the
extracellular α-dystroglycan and the transmembranelly localized
β-dystroglycan, the complex is connected to a wide variety of
proteins such as nitric oxide synthase (NOS), ion channels, kinases,
aquaporin-4, and also binds to actin through its itracellulary localized
proteins: α-dytrobrevin, syntrophins and utrophin (Culligan and
Ohlenieck, 2002; Waite et al., 2009). The interaction of DGC with this
wide variety of intracellular proteins suggests that DGC plays an
important role in the brain in different pathological conditions that
are correlated to vascular remodeling, in the same manner that
astrocytes are believed to be important in integrating signals
generated from both vessels and neurons (Lee et al., 2009).
Endothelial cells, however, may be also important in this relationship,
since they are the basis of the blood–brain barrier (Nagy et al., 2005).
The details are, however, still unknown.
Because the lack of DGC data during angiogenesis and brain
hypoperfusion we investigated the proteins β-dystroglycan, αdystrobrevin and utrophin in conjunction with alterations in laminin
and glial fibrillary acidic protein (GFAP) in both the acute phase and at
the beginning of the oligemic phase of bilateral carotid occlusion in
the rat. Another aim of our study was to measure vessel density and
blood–brain barrier disruption simultaneous with the previously
mentioned changes.
midline incision, both common carotid arteries were exposed and
carefully separated from their sheaths and vagal nerves. After
separation, arteries were doubly ligated with non-absorbable silk
sutures (3.0). The same surgical procedure was performed on the
sham-occluded group, but without the actual ligation. Sham-occluded
animal survival times were the same as for the ligated rats. Five rats
submitted to two-vessel occlusion (2VO) died after surgery.
Immunohistochemistry
Materials and methods
For histological examination animals were transcardially perfused
under ketamine/xylazine (25 and 100 mg/kg body weight, intramuscular) anesthesia with 100 mL 0.9% sodium chloride followed by
300 mL 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).
Brains were removed after perfusion and postfixed in the same
fixative for 3 days at 4 °C. Serial sections were made in coronal plane
(thickness 100 μm) by a vibration microtome (Leica VT 1000 S) in the
hippocampus and in the adjacent parietal cortex. Each section was
identified, numbered according to an atlas of rat brain (Paxinos and
Watson, 1998), and collected in phosphate buffer saline (PBS, pH 7.4).
After washing overnight in phosphate-buffered saline (PBS, 0.01 M
phosphate buffer in 0.9% NaCl, pH 7.4), floating sections were processed
for immunohistochemical reactions. Following pre-treatment with 20%
normal goat serum or 20% horse serum (α-dystrobrevin) for 1.5 h at
room temperature, the sections were incubated for 40 h at 4 °C with the
primary antibody diluted in PBS as shown in Table 1. Fluorescent
secondary antibodies (Table 2.) were used at room temperature for 3 h.
Sections were finally washed in PBS (1 h at room temperature),
mounted onto slides, coverslipped in a mixture of glycerol and
bidistilled water (1:1), and sealed with lacquer.
Pictures were taken by a BX51 microscope equipped with a DP50
digital camera (both manufactured by Olympus Optical Co., Ltd.,
Tokyo, Japan).
Experimental design
Blood–brain barrier permeability
105 adult (250–350 g) male Wistar rats were used for the study. In
one group of animals cerebral hypoperfusion was induced by
permanent bilateral carotid occlusion, whereas a sham operation was
performed in another group of animals. Ischemic and sham animals
were sacrificed on post operative day (POD) 1, 2, 4, 7, 10, 13, 17 and 30
for immunohistochemical analysis (five to six animals from each group
at every time point) and on POD 3, 7 and 30 for Western blot analysis
(four animals from each group at every time point). Components of DGC
were investigated by immunohistochemical reaction together with
laminin and glial fibrillary acidic protein (GFAP), a marker of glial
reaction in the hippocampus and in the adjacent parietal cortex.
Samples were taken from the same regions for Western blot analysis of
β-dystroglycan, utrophin, α-dystrobrevin, and laminin. To investigate
compromised blood–brain barrier permeability, rhodamine isothiocyanate (Rh IC) was used in control POD 2 and 7 rats (three animals from
each group at every time point). To measure microvascular density
intravenous ink was injected into control animals: POD 4, 7, 14, and 30
(five animals from each group at every time point). We used different
time points in Western blot analysis, blood–brain barrier permeability
and microvascular density studies that we chose after having the
immunohistochemical data from this work. All the procedures on
animals were approved by the Animal Examination Ethical Council of
the Animal Protection Advisory Board at the Semmelweis University,
Budapest and met the guidelines of the Animal Hygiene and Food
Control Department, Ministry of Agriculture, Hungary.
To investigate blood–brain barrier disruption rats were given an
intraperitoneal injection of Rhodamine isothiocyanate (RhIC, Sigma
Aldrich Ltd, Schnelldorf, Germany; 5 μL of 1% solution/g body weight)
dissolved in normal saline. RhIC was administered to two-vessel
occlusion (2VO) rats on POD 2 and 7. The rats were sacrificed 3 h after
RhIC administration (Kaur et al., 2006), then processed as above. Each
section was identified and numbered according to an atlas of rat brain
(Paxinos and Watson, 1998).
Microvascular density
After transcardial infusion with 100 mL 0.9% sodium chloride
under ketamin/xylazin anesthesia, black ink (Rotring Writing Ink,
Hamburg, Germany) was injected in the ascending aorta until the ink
Table 1
The primary antibodies applied in the study.
Surgery
Animals were anesthetized with a combination of ketamine and
xylazine (20 and 80 mg/kg body weight, intramuscular). Following a
Against
Type
Laminin
Rabbit
b
β-Dystroglycan
Mouse
a
Utrophin
β-Dystrobrevin
Mouse
Goat b
a
GFAP
Rabbit
b
Mouse
a
β-Actin
a
Firm
Dilution
for IH
Dilution
for WB
Sigma Aldrich Ltd,
Schnelldorf, Germany
Novocastra,
Newcastle-upon-Tyne, UK
Novocastra
Santa Cruz Biotechnology,
Santa Cruz, CA, USA
DakoCytomation,
Glostrup, Denmark
Sigma Aldrich Ltd
1:100
1:250
1:100
1:500
1:10
1:100
1:250
1:500
1:100
–
–
1:1500
Monoclonal. bPolyclonal. IH = immunohistochemistry, WB = Western blot.
E.A. Wappler et al. / Microvascular Research 81 (2011) 153–159
Table 2
The secondary antibodies applied in the study.
Conjugated Against Type
with
CyTM-3
Mouse
Fluorescein Rabbit
(FITC)
Fluorescein Goat
(FITC)
Firm
Donkey Jackson ImmunoResearch Laboratories
Inc., West Grove, PA, USA
Donkey Jackson ImmunoResearch Laboratories
Inc.
Donkey Jackson ImmunoResearch Laboratories
Inc.
Dilution
1:200
1:200
1:200
appeared and came out through an incision of the right ventricle
(about 1,5 mL/ 100 g body weight). The brains were then fixed by
immersion in 10% buffered paraformaldehyde for 2 days and were cut
and mounted as described above. After dehydration in an ascending
concentration series of ethanol cresyl violet counterstaining (according to Nissl) was performed.
Pictures were taken of the hippocampus and parietal cortex (3-3
on each side) on 5 parallel slices using a 20× objective. Microvascular
density was measured by an Image Pro Plus 6.0 analyzer, where black
stained vessels were measured as the percentage of the area of
interest. Each section was identified and numbered according to an
atlas of rat brain (Paxinos and Watson, 1998).
Western blot analysis
For Western blot analysis animals were transcardially perfused
with 400 mL 0.9% saline under ketamine/xylazine anesthesia. After
perfusion brains were removed, frozen in powdered dry ice, and
stored in −80 °C until further processing. The tissues of investigated
brain regions were lysed for 3 min at 100 °C in a lysis buffer composed
of 0.5 mol/L Tris (pH 6.8), glycerol, 10% SDS, 0.01% bromphenol blue,
and complete protease inhibitor (Roche Applied Science, Mannheim,
Germany). Tissue lysates were obtained by centrifugation at 12,000 ×
g for 3 min at 4 °C, and the protein concentration of each sample was
determined using the Bio-Rad protein assay kit. Equal amounts of
protein (10 μg) were subjected to electrophoresis on 10% SDS–
polyacryalamide gels. Separated proteins were then electro-transferred
to PVDF (polyvinil-difluorid) membranes (Bio-Rad). Subsequently,
primary antibodies diluted in PBS (Table 1) were added to PVDF
membranes and membranes were incubated with a mixture of
biotinylated anti-mouse/rabbit/goat immunoglobulins biotin (all from
DakoCytomation, Glostrup, Denmark) secondary antibodies. The
incubation was followed by streptavidin (DakoCytomation) secondary
antibodies-biotinylated peroxydase complex. For visualization, the ECLPlus protein detection kit was used (Amersham, Vienna, Austria)
according to the manufacturer's instructions. Band intensity was
measured using Quantity One Analysis Software (Bio-Rad). The
expression levels were calculated from specific band density and the
values were normalized to β-actin loading control.
Statistics
To analyze vascular density and Western blot results one-way
ANOVA was used followed by Tukey post hoc test. The value of p less
than 0.05 was chosen as the level of significance.
155
this increment was not as pronounced as that of laminin. In most cases
these immunoreactivities returned to control levels by POD 14, but by
the latest by POD 30. Α-dystrobrevin and GFAP immunoreactivities
did not change in the hippocampus (Fig. 1). In the adjacent parietal
cortex we observed the same changes as in the hippocampus, except
that there was an enhanced GFAP immunoreactivity which was
detected along the vessels and in every layer of the cortex, starting on
POD 2 (Fig. 1). GFAP labeling decreased later but never reached the
control level by POD 30. As reported by other investigators using this
method (Schmidt-Kastner et al., 2005) we also observed individual
variations in the overall changes. It also should be noted that changes
in laminin and DGC proteins in the cortex were “patchy” according to
the same kind of changes in neuronal death (unpublished data).
Western blot analysis
The level of β-dystroglycan decreased by POD 3 (F = 10.67; p b 0.05
vs. control) and remained low until POD 7 (p b 0.01 vs. control)
(Fig. 2), then returned to control value by POD 30. The amount of
utrophin increased significantly only by POD 7 (F = 7.53; p b 0.05 vs.
control) and maintained this level even on POD 30 (Fig. 2). However,
laminin and α-dystrobrevin expression did not change in this model
(Fig. 2). These data refer only to hippocampus, whereas no significant
change was detected to any of the examined proteins in the cortex
(Fig. 2).
Blood–brain barrier permeability and microvascular density
There was no tracer extravasation in either the hippocampus or
the cortex (data not shown). Microvascular density, however, was
elevated in both areas by POD 14 but reached statistical significance
only by POD 30 (p = 9.17; p b 0.05 vs. control) (see Fig. 3). We found
no differences between layers of the hippocampus, such as the
stratum oriens, stratum pyramidale and stratum radiatum, or
between any of the cortical layers. However, the increased vascular
density in the cortex showed a patchy pattern, that “patches” usually
included every layer.
Discussion
Cerebral hypoperfusion had a notable impact on the immunoreactivity of laminin, some of the DGC proteins in the hippocampus,
on the adjacent cerebral cortex, and on cortical GFAP after bilateral
carotid occlusion. All the investigated changes except GFAP, returned
to baseline by the 14th to 30th day of the brain hypoperfusion and at
the same time a denser vascular network was observed. During all
these changes the blood–brain barrier remained intact, which is
consistent with previous observations in this model (de Wilde et al.,
2002; Farkas et al., 2007). One possible reason is the relatively mild
ischemia following the occlusion, which is thought to be insufficient
to harm the isolating function of the blood–brain barrier (BBB)
(Farkas et al., 2007). However, ultrastructural abnormalities, such as
basement membrane thickening caused by fibrous collagen deposits,
could be detected 14 months after the onset of bilateral carotid
occlusion, but not after 13 weeks (de Jong et al., 1999; de Wilde et al.,
2002; Farkas et al., 2007).
Immunohistochemistry
Alterations in β-dystroglycan and laminin immunoreactivities, as
markers of glio-vascular decoupling after chronic bilateral carotid
occlusion
We found an early (POD 2) decrease in β-dystroglycan immunoreactivity with an enhanced immunoreactivity of laminin in the dorsal
and ventral hippocampus (see Fig. 1). The most prominent changes in
the immunoreactivities were detected between POD 4 and 7.
Utrophin immunoreactivity also increased by POD 7 (Fig. 1); however
The loss of the β-dystroglycan immunopositivity or the increase of
the laminin immunoreactivity occurs in other brain injury models
using adult animals as well: autoimmune encephalitis, systemic
venom injection, cerebral stab wound, focal brain ischemia; and
sometimes are not accompanied by BBB opening (Agrawal et al.,
Results
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E.A. Wappler et al. / Microvascular Research 81 (2011) 153–159
Fig. 1. Representative photos of laminin, β-dystroglycan, utrophin, α-dystrobrevin and glial fibrillary acidic protein (GFAP) immunoreactivities in the rat hippocampus and cortex at
differet time points after the onset cerebral hypoperfusion (scale bar = 100 μm). POD = post operative day.
2006; Rapôsoa et al., 2007; Szabo and Kalman, 2008; Milner et al.,
2008; Del Zoppo and Mabuchi, 2003; Hamann et al., 1996; Wagner
et al., 1997; Velkamp et al., 2005b). The results of this and other
studies, in contrast to some previous findings, indicate that under
different pathological conditions glio-vascular decoupling occurs
rather than increased BBB permeability. In addition, our observation
that remarkable changes in the immunoreactivity of laminin was not
accompanied by any change in its expression supports the findings
that changed laminin immunoreactivity was not a sign of increased
tracer extravasation through the BBB (Rapôsoa et al., 2007; Szabo and
Kalman, 2008). Previous studies have also confirmed that the visibility
of laminin in the brain depends on its accessibility for the antibody,
which can change dramatically when the glio-vascular connection is
newly formed or when the vessels are injured (Krum et al., 1991;
Szabo and Kalman, 2004; Zand et al., 2005). Surprisingly, in this
model, elevated matrix metalloproteinase activity, which is important
in different vascular remodeling was only described in the white
matter (Ihara et al., 2001; Nakaji et al., 2006; Sood et al., 2009). It
should be noted here that in our work no change in expression was
detected in the cortex in any of the examined proteins, but this is most
likely because it is less affected by hypoperfusion in this model and
because of the patchy pattern of the cortical changes that occur
following permanent bilateral carotid occlusion.
Urophin, but not β-dystroglycan has prolonged expression after chronic
bilateral carotid occlusion
In our model the increase in utrophin immunoreactivity started
only 5 days later than that of laminin and β-dystroglycan, but
returned to control level in synchrony with both laminin and βdystroglycan. In the hippocampus its expression also increased with
the observed immunohistochemical changes and was still elevated on
the 30th day after the onset of bilateral carotid occlusion, when a very
low, baseline immunoreactivity was seen. This provides two observations: (1) The detectibility of this intracellulary localized protein is
more difficult with immunohistochemistry during glio-vascular
E.A. Wappler et al. / Microvascular Research 81 (2011) 153–159
157
Fig. 2. (A) Representative blots of laminin, β-dystroglycan, utrophin and α-dystrobrevin from the hippocampus and the parietal cortex 3, 7 and 30 days after the onset of cerebral
hypoperfusion. (B) Expression of laminin, β-dystroglycan, utrophin and α-dystrobrevin 3, 7 and 30 days after the onset of cerebral hypoperfusion in the hippocampus.
(C) Expression of laminin, β-dystroglycan, utrophin and α-dystrobrevin 3, 7 and 30 days after the onset of cerebral hypoperfusion in the cortex. Optical densities of bands are
expressed as percentages of the control levels (sham operated animals). Data are expressed as means ± SEM. *p b 0.05, **p b 0.001 vs. control. POD = post operative day.
decoupling than laminin (its expression is almost significant by
POD3). (2) Utrophin probably has other, vessel-related compensatory
mechanisms during cerebral hypoperfusion, since utrophin is also
highly expressed in brain microvessel endothelial cells (Culligan and
Ohlenieck, 2002; Haenggi and Fritschy, 2006), in contrast to laminin
or β-dystroglycan. The other intracellular DGC protein investigated in
our study, α-dystrobrevin, remained almost unchanged, only a slight
decrease was detected in its expression and immunoreactivity during
the ischemic phase of brain hypoperfusion. This can be related to its
different tissue distribution and less important role in this brain
pathology compared to utrophin.
Reactive gliosis occurs only in the cerebral cortex during the 30-day
examination period in our brain hypoperfusion model
We also investigated reactive gliosis in correlation with DGC and
laminin alterations. Glial cells are important in supporting neurons
during brain hypoperfusion. On the other hand, glial cells are strongly
associated with DGC and laminin around the vessels and in this way
they can help vascular remodeling by forming a “skeleton” for
vascular rearrangements and also by expressing VEGF (Culligan and
Ohlenieck, 2002; Schmidt-Kastner et al., 2005; Perrin et al., 2009).
Previous studies (Schmidt-Kastner et al., 2005; Institoris et al., 2007)
support our results that no change in the hippocampus can be
observed in GFAP immunoreactivity during this period, but a robust
increment in the cortex can be detected starting very early during
brain hypoperfusion and lasting beyond day 30, the last day of
observation of the experiment. The multiple roles of glial cells in brain
hypoperfusion may explain the sustained gliosis. The unchanged
hippocampal GFAP immunoreactivity in this model is probably due to
its initially high expression in this region (Kálmán and Hajós, 1989;
Zilles et al., 1991), where significant elevation is harder to detect. The
unchanged immunoreactivity was, however, correlated to sham
controls in this study with the same post operative survival, while
2VO itself also can slightly increase the GFAP density in the
hippocampus (Institoris et al., 2007).
The observed changes may be significant in cerebral hypoperfusion
related angiogenesis
Our results, with the increased number of brain vessels in our
model, show that the changes in laminin, β-dystroglycan, and
utrophin immunoreactivities are strongly associated with the process
of vascular remodeling. Although there have been researchers with
other findings (de Wilde et al., 2002; Schneider et al., 2007), our
results are consistent with previous observations by Ohtaki et al.
(2006) and Sekhon et al. (1997) who found elevated numbers of brain
vessels using this model. Moreover, following bilateral carotid
occlusion an augmented VEGF expression was reported between
POD 14 and 30 (Ohtaki et al., 2006). If we further scrutinize the link
between the observed changes in extracellular matrix to angiogenesis
in this model, we find that both eNOS and nNOS levels are altered in
the examined regions until POD 7 (de la Torre et al., 2003; Mracsko
et al., 2010), which are important triggers of angiogenesis (Ziche and
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E.A. Wappler et al. / Microvascular Research 81 (2011) 153–159
Fig. 3. (A) Representative photos of hippocampal and cortical vasuclar densities 7, 14 and 30 days after the onset of cerebral hypoperfusion. (B) Quantitative analysis of vascular
densities in the hippocampal CA1, CA2-3, gyrus dentatus regions and in the cortex , 14 and 30 days after the onset of cerebral hypoperfusion (scale bar = 100 μm). Data are expressed
as means ± SEM. *p b 0.05, **p b 0.001 vs. control. POD = post operative day.
Morbidelli, 2000; Beaudry et al., 2009). The timing of all the observed
changes together with the fact that nNOS is related to DGC
intracellulary (Culligan and Ohlenieck, 2002) confirm and support
our conclusion of an important relationship between DGC and laminin
changes, and angiogenesis following cerebral hypoperfusion.
Conclusions: Possible role of DGC and laminin in
cerebral angiogenesis
The main finding of this study is that in our model of cerebral
hypoperfusion alterations in the immunoreactivities and expressions
of β-dystroglycan, utrophin, and laminin seem to correlate with
angiogenesis rather than BBB disruption. Utrophin, however, may
play an important role in further, vessel-related compensatory
mechanisms.
Acknowledgments
This work was supported by OTKA 60930 (OTKA holder: Professor
Mihály Kálmán, MD, PhD, DSc). The authors would like to thank
Andrea Őz and Szilvia Deák for their excellent technical assistance and
special thanks Nancy Busija, MA, for critical reading and editing this
paper. We also would like to thank Miklós Mózes, MD, PhD and Gábor
Kökény, MD, PhD for the Image Pro Plus 6.0 analyzer.
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