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Expression of nestin in H-Tx rats with inherited congenital hydrocephalus

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Title:
Expression of nestin in H-Tx rats with inherited congenital hydrocephalus
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Somera, Kathleen
Jones, Hazel ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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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.,


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



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.


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.


Immunohistochemistry






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.



Table 1

Distribution of Nestrin Immunoreactivity and Histological Observations

Brain
Secti on Hydrocephalic Normal

Lateral
Overexpression
ventricle
of nestin, Nestin immunoreactivity not as intense
(neuroepithelial
flattened cells
lining)


Overexpression

in fibers,

Lateral increased

ventricle intercellular
Nestin immunoreactivity not as intense, cells intact, no increased intercellular spaces, no sign of disorganization
(subventricular spaces, and

zone) disorganization

of cells and

fibers


No
Aqueduct roof Intense staining of fibers
Immunoreactivity






Intense staining of fibers


RESULTS



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.

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.
















p.,
di

4
I'


�i', *


Some

Aqueduct floor immunoreactivity

of fibers


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.



DISCUSSION



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.






ACKNOWLEDGEMENTS



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.


REFERENCES






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.

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hydrocephalus in H-Tx rats. Child's Nervous System. 16: 578-584.

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8. Takano T, Rutka JT, Becker, LE (1996) Overexpression of nestin and vimentin in ependymal cells in

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