Journal of Undergraduate Research
Volume 4, Issue 7 - March 2003
Expression of nestin in H-Tx rats with inherited congenital hydrocephalus
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.
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.,
UP Journal of Undergraduate Research University of Florida
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
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.
Fixation and Tissue Preparation
Rat pups were intraperitoneally injected with 0.05 ml sodium pentobarbital (Ig/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%/o (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 immunoglobulinl: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).
Analysis of Results
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.
Distribution of Nestrin Immunoreactivity and Histological Observations
Secti on Hydrocephalic Normal
of nestin, Nestin immunoreactivity not as intense
Nestin immunoreactivity not as intense, cells intact, no increased intercellular spaces, no sign of disorganization
(subventricular spaces, and
of cells and
Aqueduct roof Intense staining of fibers
Intense staining of fibers
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 O/% cresyl violet.
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.
Aqueduct floor immunoreactivity
Figure 2. Brain sections showing nestin immunoreactivity. Sections were counterstained with nissi
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.
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.
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.
1. Boillat CA, Jones HC, Kaiser GL (1999) Aqueduct stenosis in hydrocephalus: ultrastructural investigation in
neonatal H-Tx rat brains. European Journal of Pediatric Surgery. 9:44-46.
2. Jones HC, Lopman BA, Jones TW, Carter BJ, Depelteau JS, Morel L (2000) The expression of inherited
hydrocephalus in H-Tx rats. Child's Nervous System. 16: 578-584.
3. Matsuda M, Katoh-Semba R, Kitani H, Tomooka Y (1996) Possible role of the nestin protein in the developing
central nervous system in rat embryos. Brain Research. 723: 177-189.
4. Nojima Y, Enzan H, Hayashi Y, Nakayama H, Kiyoku H, Hiroi M, Mori K (1998) Neuroepithelial and ependymal
changes in H-Tx rats with congenital hydrocephalus:An ultrastuctural and immunohistochemical study. Pathology
5. Overholser MD, Whitley JR, O'Dell BL, Hogan AG (1954) The ventricular system in hydrocephalic rat brains
produced by a deficiency of vitamin B12 or folic acid in the maternal diet. Anatomical Record 120:917-933.
6. Rodriguez E, Rodriguez S, Hein S (1998) The subcommissural organ. Microscopy and Technique. 41:98-123.
7. Rodriguez S, Vio K, Wagner C, Barria M, Navarrete E, Ramirez V, Perez-Figares J, Rodriquez E (1999) Changes in
the cerebrospinal-fluid monoamines in rats with an immunoneurtralization of the subcommissural organ-Reissner'
fiber complex by maternal delivery of antibodies. Experimental Brain Research. 128:278-290.
8. Takano T, Rutka JT, Becker, LE (1996) Overexpression of nestin and vimentin in ependymal cells in
hydrocephalus. Acta Neuropathology. 92: 90-97.
9. Tohyama T, Lee V, Rorke L, Marvin M, McKay R, Trojanowski J (1992) Nestin expression in embryonic human
neuroepithelium and in human neuroepithelial tumor cells. Laboratory Investigation. 66:303.
10. Vio K, Rodriguez S, Navarrette E, Perez-Figares J, Jimenez A, E Rodriguez (2000). Hydrocephalus induced by
immunological blockage of the subcommissural organ-Reissner's fiber complex by maternal transfer of anti-RF
antibodies. Experimental Brain Research. 135:41-52.
Back to the Journal of Undergraduate Research
College of Liberal Arts and Sciences I University Scholars Program I University of Florida |I UNIVERSITY of
'Pi, /Universii of Fr NC A 3"(2 8 0 .
University of Florida, Gainesville, FL 32611; (352) 846-2032.