Journal of Undergraduate Research
Volume 4, Issue 7 - March 2003
Expression of nestin in H-Tx rats with inherited congenital hydrocephalus
Kathleen Somera
ABSTRACT
Hydrocephalus occurs in 0.5-1.5 per 1000 births. It is a neurological
condition that results from the accumulation of excess cerebrospinal
fluid (CSF). Despite substantial research, the pathogenesis of hydrocephalus
is not well understood. The present study aims to determine abnormalities
in lateral ventricles and cerebral aqueduct in a rat strain with inherited
hydrocephalus associated with aqueduct stenosis and lateral ventricle
dilatation. Immunohistochemistry was used to investigate the distribution
of nestin, an intermediate filament protein, in brain from newborn H-Tx
and Sprague Dawley rat brains. Hydrocephalic H-Tx rats, characterized
by dilated lateral ventricles, showed overexpression of nestin in the
neuroepithelial lining of lateral ventricles. In the subventricular
zone, immunoreative fibers appeared disorganized, and intercellular
spaces were expanded. Normal brains from both strains also expressed
nestin in the neuroepithelial lining and subventricular zone but with
reduced intensity and distribution.
The results suggest that nestin is a good marker for neuroepithelial
disruption in congenital hydrocephalus. Furthermore, the absence of
immunoreactive cells in the aqueduct roof strongly indicates subcommissural
organ (SCO) involvement in hydrocephalus pathogenesis. In the stenosed
aqueduct of hydrocephalic brains, there was a complete absence of nestin
staining in the roof. In contrast, normal brains had marked immunoreactivity.
The roof of the aqueduct is the site of the SCO, which appeared to be
absent in hydrocephalic rats. The floor of the aqueduct contained nestin
staining in both groups, but it was less dense in the hydrocephalic
rats. Future studies will monitor the progression and morphological
changes throughout early stages of congenital hydrocephalus with the
objective to learn more about its pathogenesis.
INTRODUCTION
Hydrocephalus occurs in 0.5-1.5 per 1000 births. It is a neurological
condition that occurs as a result of accumulation of excess cerebrospinal
fluid (CSF), the clear fluid that surrounds the brain and spinal cord.
CSF protects the brain from injury and maintains chemical homeostasis.
The imbalance of CSF production and absorption can cause abnormal accumulation
in the brain. Surgical treatment of hydrocephalus requires insertion
of a shunt to drain excess CSF. Despite shunt treatment, neurological
deficits including motor abnormalities and psychiatric disorders persist
in treated patients. In addition, infection and obstruction complicate
shunt treatment. As a result, researchers are seeking other methods
of treatment.
Studies in H-Tx rats with inherited congenital hydrocephalus showed
that ventricular dilatation is associated with aqueduct stenosis, characterized
by an abnormally reduced aqueduct lumen (Boillat et. al., 1999). Investigators
have reasons to believe that ventricular dilatation and aqueduct stenosis
may be outcomes of a chain of biochemical processes that originate during
fetal brain development (Nojima et. al., 1998; Boillat et. al., 1999).
Nojima points out changes in ependyma and neuroepithelium of the lateral
ventricles and aqueduct, and Boillat concludes that there may be a direct
correlation between ventricular dilation and ependymal effect. Earlier
studies have dealt with rats after birth, and only few reports on fetal
studies are available. The present study aims to examine the early stages
of hydrocephalus through careful analysis of expression of cell-specific
antigens like nestin in newborn rats. The study of cell-specific antigens
such as nestin early in the disease may provide clues as to which cells
are abnormal, particularly in the lateral ventricle and aqueduct regions.
The objective of this study is to compare the immunoreactivity of brain
cells in Sprague Dawley (SpD) and H-Tx rats to determine which cells
in dilated lateral ventricles and aqueduct regions are abnormal.
Nestin proteins are especially abundant in the neuroepithelial stem
cell of the rat. Nestin is a type VI intermediate filament (IF) protein,
a type of cytoskeletal protein (Tohyama et. al., 1992). Nestin expression
occurs during early developmental stages and during regenerative processes
in neuronal and muscle cells. However, nestin is developmentally regulated
and its expression in rats decreases at eleven-days gestation and is
extinguished by postnatal day six (Tohyama et. al., 1992). According
to Matsuda and colleagues (1996), nestin was first detected in the developing
rat central nervous system (CNS) as an epitope expressed in neuronal
precursors but not by mature neurons.
MATERIALS AND METHODS
Animals
The H-Tx rats maintained at the University of Florida were obtained
from inbred pairs provided in 1992 by D. F. Kohn, Columbia University,
New York. Brother-sister mating of normal rats except for occasional
cousin matings maintained the colony. Sprague-Dawley rats were obtained
from Dr. DeMarco’s laboratory at the University of Florida, Gainesville,
Florida. Zero day hydrocephalic and control H-Tx and Sprague Dawley
rats were used in immunohistochemisty. Rats immediately taken after
birth were designated zero day rats. All H-Tx rats may have abnormal
genotypes, but because the expression of hydrocephalus is complex, not
all H-Tx rats are hydrocephalic (Jones et al., 2000). As a result, Sprague
Dawley rats, a non-hydrocephalic strain, were also studied as controls.
Zero-day rats were used to ensure focus on early stages of hydrocephalus.
A total of nine animals were used: four hydrocephalic H-Tx rats with
ventricle dilatation, two normal H-Tx rats, and three normal SpD rats.
Hydrocephalic animals were identified at birth by enlarged, “domed”
heads.
Rat pups were intraperitoneally injected with 0.05 ml sodium pentobarbital
(1g/ml) (Vet. Lab, Inc., Lexesa, Kansas) and perfused transcardially
with 0.9% saline followed by fixative containing 4% paraformaldehyde
in 0.1 M phosphate buffer, pH 7.3. Brains were excised carefully and
postfixed in 4% paraformaldehyde for 2 h at room temperature. They were
cryoprotected overnight in 15% (15g/100ml) and 30% (30g/100ml) sucrose
in phosphate buffered saline (PBS) at 4… C. Following cryoprotection,
brains were frozen in isopentane (-30… C to -50… C) and carefully wrapped
to prevent dehydration. They were stored at -80 0 C until sectioned.
Coronal 20 mm thick sections were prepared on a cryostat (Microm HM
505 E). Sections 200 mm apart were dried onto slides (Fisher Superfrost
Plus) and stored at -80… C.
Slides with sections showing aqueduct and lateral ventricles were chosen
for immunostaining. These slides represented the posterior and anterior
forebrain, respectively. The presence of aqueduct stenosis and lateral
ventricle dilatation confirmed hydrocephalus in affected rats. For every
staining run, control and hydrocephalic rat brain tissues from the same
age groups were stained simultaneously for comparisons. Following rehydration
of tissue sections in PBS and distilled water, they were treated with
0.1% hydrogen peroxide for 30 min. Sections were incubated for 1 h with
non-specific staining blocking solution, 10 % normal horse serum in
1% bovine serum albumin (BSA) in PBS. Sections were incubated with 1:75
nestin primary antibody in 1% BSA in PBS overnight. Antiserum against
Nestin, a purified mouse anti-rat nestin monoclonal antibody, was purchased
from BD Pharmingen International. To prove specificity, tissues incubated
in the antibody diluent alone, 1% BSA in PBS, were used as negative
controls. After several washes with PBS, sections were incubated for
1 h with biotinylated horse anti-mouse immunoglobulin1:400 dilution
in 1% BSA in PBS. Antibody reaction was visualized by incubating tissues
with a solution of avidin-biotin complex (ABC) (Elite Vectastain Kit,
Vector Laboratories Inc., Burlingame, CA) for 1 h and developed using
diaminobenzine. Sections were dehydrated in 70%, 95% and 100% ethyl
alcohol and cleared in xylene for 5 min. Coverslips were mounted with
D.P.X. (Aldrich, Milwaukee, WI).
All slides were examined under a light microscope. The immunostained
slides were examined for staining differences. In addition, nestin slides
were counterstained with 0.1% cresyl violet to visualize anatomical
details. Slides were also compared to adjacent cresyl violet slides
to aid in cell and regional identification.
The Nestin immunoreactivity, shown by dark brown staining, was predominantly
expressed in neuroepithelial lining of lateral ventricles, subventricular
zone, and aqueduct (Table 1). All four hydrocephalic
H-Tx rats, characterized by dilated lateral ventricles, showed overexpression
of nestin in the neuroepithelial lining (Fig. 1, Fig. 2A).
Cells at this site showed flattening. In the subventricular zone, immunoreative
fibers appeared disorganized and disoriented. In addition, the subventricular
zone showed widening of intercellular spaces. Normal brains also expressed
nestin in neuroepithelial lining and subventricular zone but with reduced
intensity and distribution (Fig. 2B). Unlike hydrocephalic
brains, cells and fibers of the subventricular zone remained intact
and organized in normal brains. All the normal brains, regardless of
strain, showed the same immunoreactivity. Figure 1. Dilated lateral ventricles confirm the
presence of hydrocephalus in newborn rats. A: H-Tx rat brain with
severe hydrocephalus. B: H-Tx rat brain with mild hydrocephalus. C:
Normal SpD brain. Arrows point to lateral ventricles. Brain sections
were stained with 0.1 % cresyl violet. 400x magnification. Fibers around the cerebral aqueduct also
showed immunoreactivity. The fibers of the aqueduct floor in hydrocephalic
brains showed immunoreactivity, but the roof totally lacked nestin expression.
In contrast, fibers of both the floor and roof of the aqueduct showed
immunoreactivity in normal brains regardless of strain. In addition,
aqueduct fiber immunoreactivity in normal brains was more intense than
in hydrocephalic brains (Fig. 2 D, F, G, and H).
The roof of the aqueduct consists of cells forming the subcommissural
organ (SCO), which was absent in hydrocephalic brains. Figure 2. Brain sections showing nestin immunoreactivity.
Sections were counterstained with nissl to identify cells. Immunostaining
of the neuroepithelial lining of lateral ventricles of hydrocephalic
brains showed flattening of cells and widening of intercellular spaces
as indicated by the asterisk (A). In normal brains, nestin was also
expressed in the neuroepithelial lining, but with less intensity (B).
Note the presence of cells and absence of intercellular spaces (B).
Hydrocephalic brains showed immunoreactivity in aqueduct floor fibers
(E) but not in aqueduct roof fibers (C). Normal brains showed immunoreactivity
in both the floor and roof of the aqueduct (D and F, normal H-Tx rats;
G and H, normal SpD rats). Arrows indicate nestin immunoreactivity.400x
magnification.
Nestin immunoreactivity was examined to determine any existing abnormality
in hydrocephalic cytoskeletal filament distribution and cell behavior.
Nestin immunoreactivity showed significant differences between normal
and hydrocephalic rats. I am grateful for Dr. Hazel C. Jones for her assistance
and support as a mentor and for Dr. De Marco for supplying Sprague Dawley
rats. Back to the Journal of Undergraduate
Research © University of Florida,
Gainesville, FL 32611; (352) 846-2032.
Fixation and Tissue Preparation
Immunohistochemistry
Analysis of Results
Table 1
Distribution of Nestrin Immunoreactivity and Histological Observations
Brain Section
Hydrocephalic
Normal
Lateral ventricle
(neuroepithelial lining)Overexpression of nestin, flattened cells
Nestin immunoreactivity not as intense
Lateral ventricle
(subventricular zone)Overexpression in fibers, increased intercellular spaces, and
disorganization of cells and fibers
Nestin immunoreactivity not as intense, cells intact, no increased
intercellular spaces, no sign of disorganization
Aqueduct roof
No immunoreactivity
Intense staining of fibers
Aqueduct floor
Some immunoreactivity of fibers
Intense staining of fibers
RESULTS
DISCUSSION
In hydrocephalic brains, nestin was expressed in the floor of the aqueduct,
neuroepithelial lining of the lateral ventricles, and fibers in the
subventricular zone. This result was consistent with a study of nestin
and vimentin overexpression in rats by Takano and colleagues (1996).
According to Takano, nestin was predominantly expressed in neuroepithelial
cells and radial glial fibers during neuronal migration. Hydrocephalic
brains overexpressed nestin in cells of ependymal disruption. This result
can be explained by the loss and disruption of ependymal cells in dilated
ventricles of hydrocephalic brains as indicated by Takano. Studies have
found that nestin protein is overexpressed in areas of cell disorganization
and abnormality. For example, Tohyama and colleagues (1992) concluded
that nestin appeared to be more abundant in the least mature gliomas
that show most malignant behavior. As mentioned in the introduction,
nestin is developmentally regulated and its expression in rats decreases
from eleven-days gestation and extinguished in postnatal day six (Tohyama
et. al., 1992). The results in this study and past studies suggested
that the overexpression of nestin is related to cell disorganization
and disruption in hydrocephalus. Matsuda and colleague’s study
of nestin in rat CNS (1996) suggested a possible role for nestin: maintenance
of neuroepithelial cells. Thus, the presence of nestin can be used as
a marker for cell damage.
Since the aqueduct roof fibers of hydrocephalic brains lacked immunoreactivity,
they might be directly involved in hydrocephalus. The lack of immunoreactivity
in the aqueduct roof may be due to the absence of the SCO in the occluded
aqueduct of hydrocephalic H-Tx rats used in this study. The SCO secretes
glycoproteins to form Reissner’s fiber (RF), which grows along
the cerebral aqueduct, fourth ventricles, and central canal of the spinal
cord (Rodriguez, et. al., 1999). Therefore, without the SCO, RF does
not form, and thus nestin immunoreactivity is not observed. Furthermore,
the SCO may play a major role in hydrocephalus. The primary function
of the SCO in neurological processes is unknown, but some evidence suggests
that it may participate in the circulation of CSF (Rodriguez et. al.,
1998). Recent studies support the SCO hypothesis proposed by Overholser
(1954) that a dysfunction of the SCO leads to aqueduct stenosis and
hydrocephalus. Vio and colleagues (2000) found that permanent absence
of normal RF in rats followed by aqueduct stenosis caused hydrocephalus
(2000). In addition, immunocytochemical studies of the SCO with hydrocephalus
in rats showed signs of SCO size reduction (Boillat et. al., 1999).
Hydrocephalic rats used in the present study also had occluded aqueduct
and lacked an SCO.
Results suggested that the expression of nestin is related to hydrocephalus
and its characteristic neuroepithelial cell disruption and aqueduct
stenosis. Nestin immunoreactive cells were abnormal in the dilated ventricles
and in the aqueduct region of hydrocephalic rats when compared to normal
rats. The effect and extent of their immunoreactivity at this point
is hard to tell, but further examination in fetal brains can confirm
the findings of this study. The study of specific cell antigens early
in the disease may provide clues as to which cells are abnormal, particularly
in the lateral ventricle and aqueduct regions, and their reactivity
can be monitored throughout developmental progression. In addition to
nestin, vimentin, an intermediate filament protein, should also be studied
in fetal brains. Vimentin, in a past study, also showed significant
differences in immunoreactivity in different animals, particularly in
neuroepithelial lining of the lateral ventricles. According to Takano
and colleagues (1996), nestin and vimentin are co-localized at some
point in neuronal development. Study of these cell-specific antigens
could provide clues as to what causes hydrocephalus in H-Tx rats. In
addition, further examination of the SCO and RF changes in rat brains
may also give a better understanding of hydrocephalus and its pathogenesis.
ACKNOWLEDGEMENTS
REFERENCES
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