Pathology International 1998; 48: 11 5-1 25

Original Article Neuroepithelial and ependymal changes in HTX rats with congenital hydrocephalus: An ultrastructural and immunohistochemical study

Yuji Nojima,' Hideaki Enzan: Yoshihiro Hayashi: Hirofumi Nakayama: Hiroshi Kiyoku: Makoto Hiroi: and Koreaki Mori' Departments of Neurosurgery and 2Pathology, Kochi Medical School, Kochi, Japan

To Investigate the pathogenesis of Congenital hydro- cephalus the brains of HTX rats aged between 16 days and 4 weeks and the brains of normal Wistar rats of the same ages were examined. In the fetal HTX rat brains, the lateral ventricles were symmetrically dilated from 20 days of gestation. The neuroepithelium bordering the ventricles showed thinning with cellular disarrangement and deformity. Similar neuroepithelial abnormalities were also found in the lateral Ventricles of the HTX rat brain with no macroscopic slgns of hydrocephalus at 20 days of gestation. The neuroepithelium showed flattening of the cells, widening of the Intercellular spaces, formation of microvilli on the detached lateral cell surfaces, and frequent macrophage infiltration. On the other hand, the neuroepithelial cells of the third ventricle and the aqueduct were affected less severely or showed no signlficant abnormalities. lmmunohistochemlcally, most of the neuroepithelium and ependyma of the lateral ventricles were positive for vimentin in both prenatal and postnatal hydrocephalic HTX rats, while a small number or none of those in normal control rats were positive. These morphological changes suggested that Preferential involvement of the lateral ventrlcular neuroepithelium might be closely associated with the pathogenesis of congenital hydrocephalus in HTX rats.

Key words: congenital hydrocephalus, electron microscopy, ependyma, HTX rat, neuroepithelium

Congenital hydrocephalic HTX rats are an animal model of hydrocephalus that were bred from albino rats by Kohn etal.' Hydrocephalus occurs in these rats with an incidence of

Correspondence: Yuji Nojima, MD, Department of Neumsurgery, Kochi Medical School, Kohasu, Okohcho, Nankoku-city, Kochi 783, Japan. Email: <nojimay@Kochi-ms.ac.jp>

Received 15 April 1997. Accepted for publication 22 September 1997.

approximately 40% after brothersister mating, and the mode of inheritance is assumed to be polygenic.l** However, to date the pathogenesis of hydrocephalus in HTX rats has not been established. Poorly developed veins in the periostealdural layers and undeveloped pia-arachnoid cells,' as well as aqueductal closure due to abnormal brain development of the midline region of the dorsal mesencephalon,3 have been reported to have a role in its pathogenesis. However, Wada has not detected such abnormalities and suggested that they were secondary changes caused by advanced hydrocephalus?

While hydrocephalus in HTX rats has been reported to occur from 18 to 20 days of gestation? most earlier studies have dealt with rats just after and only a few reports on fetal s t~d ies~* '~ are available. Furthermore, systematic ultrastructural examination of fetal HTX rats has not yet been reported. We consider that the examination of morphological changes at the early stage of congenital hydrocephalus is essential for clarifying the pathogenesis. In particular, changes of the ventricular ependyma are closely related to the severity or progression of hydrocephalus, although the ventricular ependyma varies depending on lacation,"-" and the changes occurring in hydrocephalus are not u n i f ~ r r n . ~ ~ ~ ~ ~ ~ ~

Using light and electron microscopy, as well as immunohistochemistv, in the present study we observed neuroepithelial and ependymal changes lining the lateral ventricle, the third ventricle, and the aqueduct of HTX rats from late gestation to the early postnatal period. In a rat with onset of hydrocephalus in late gestation, the neuroepithelium of lateral ventricles showed more severe changes compared to that located in any other ventricular system. This is the first report concerning the prenatal preferential neuroepithelial lesions of the lateral ventricles in hydrocephalic HTX rats.

1 16 Y. Nojima eta/.

MATERIALS AND YETHODS

Animals

Mx rats established by Kohn ef a/.’2 were subsequently inbred by brothersister mating. All animals were fed a C€-2 diet (CLEA Japan Inc., Tokyo, Japan]. A mature female HTX rat was mated overnight with a male HTX rat. On the following morning, vaginal smears were obtained and female rats showing sperm-positive smears were considered to be at day 0 of gestation. Pregnant females were killed on days 16, 18, and 20 of gestation. The fetuses were removed from the uterus and killed by decapitation. Normally delivered HTX rats were also killed by decapitation with ether anesthesia at the age of 1 day, as well as 1,2,3, and 4 weeks af€er birth.

Atotal of 60 HTX rats were ueed. Among them 22 rats were macroscopically hydrocephalic, including one rat at the age of 20 days of gestation, six rats at 1 day after birth, and six, three, three, and three rats at 1,2,3, and 4 weeks after birth, respectively. The remaining 38 HTX rats were non- hydrocephalic (seven, seven, and ten rats at the age of 16, 18, and 20 days of gestation, respectively; four 1-day-old rats; and four, two, two, and two rats at, 1,2,3, and 4 weeks after birth, respectively; Table 1 ).Among them 42 brains were processed for light microscopy and imrnunohistochemistry, and 18 brains for electron microscopy. In addition, 40 Wistar rats (CLEA) matched for age (five rats at each age) were used as normal controls. Among them 24 brains were examined by light microscopy and immunohistochemistry and 16 brains (two rats at each age) were also used for electron microscopy.

Ught microscopy

The brains of rats from 16 days of gestation to 4 weeks after birth were fixed with 10% phosphate-buffered formalin and

then cut coronally from the frontal lobe to the brain stem. The tissues were dehydrated with a graded ethanol series and embedded in paraffin wax. Paraffin sections (4pm thick) were stained with hematoxylin and eosin (HE) and then mounted in Eukitt (Kindler, Freiburg, Germany).

lmmunohistochemistry

Deparaffinized and hydrated sections of formalin-fixed and paraff in-embedded brain tissues were processed for the blocking of endogenous peroxidase and non-specific staining. The sections were incubated for 1 h at room temperature with mouse monoclonal antibodies (mAb) against vimentin (the mAb, clone V9, diluted I : 50; Dako, Glostrup, Denmark). After rinsing with 0.01 mollL phosphate- buffered saline (PBS; pH 7.2), the sections were incubated for 30 min at room temperature with biotinylated rabbit anti- mouse IgG + IgA + IgM (Histofine SAB-PO(M) kit; Nichirei, Tokyo, Japan), followed by incubation with streptoavidin- peroxidase solution (Histofine SAB-PO(M) kit) for 30 min at room temperature. Peroxidase activity was visualized by incubation in 0.05moVL Tris buffer (pH 7.6) containing 0.02% 3,3’diaminobenzidine tetrachloride (Dojin, Kumamoto, Japan) and 0.01% hydrogen peroxide. Finally, the sections were counterstained with hematoxylin and mounted in Eukitt. Control stainings without primary antibody with non-immune mouse IgG fractions were negative.

Transmission electron microscopy

The sites of tissue sampling were the dorsal wall of the anterior horn overlying the corpus callosum and the lateral wall of the anterior horn overlying the caudate nucleus in the lateral ventricle, the posterior horn of the lateral ventricle, the

Table 1 Summary of morphological findings in the lateral ventricle and lining neuroepithelium and ependyma of HTX rats at various ages

Age (no. rats) Ventricular dilatation Salient morphological findings

GI€d (n = 7) - (om None G18d (n = 7) - ( o m None G2W (n = 11) + ( l / l l )

- (10111) Thinning of neuroepithelium; cellular disarrangement and deformity One of them showed similar changes to those in a hydrocephalic rat, but no abnormalities in other

Widening of intercellular space; cellular disarrangements and deformity; increase of 9 rats.

microfilaments Pld (n = 10) ++ (6/10)

- (4t10) None P7-28d (n = 25) +++ (1925) Decrease of cilia and microvilli; flattened ependymal cell; increase of intermediate filaments

(these changes increased in severity with age) - (10/25) None

Total (n = 60). Ventriculardilation: (-) none; (+) mild; (++) moderate; (+++) severe. G, gestational; P, postnatal; d, days.

Ependyma in congenital hydrocephalic rat 117

third ventricle, and the aqueduct. Specimens from each site were prefixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH7.4) for 2h at 4OC, and then postfixed in 1% osmium tetroxide in the same buffer for 2h at 4OC. The specimens were subsequently dehydrated with an ethanol series and embedded in epoxy resin. Semithin sections (about 1 pm thick) were stained with rnethylene blue and azure II solution and used for the selection of appropriate areas for ultrathin sectioning. The ultrathin sections were stained with uranyl acetate and lead citrate, and were viewed with a transmission electron microscope (JEM, 100s; JEOL, Tokyo, Japan).

RESULTS

Light microscopic findings

In all rats at 16 days of gestation the ventricular system was open and lined by a pseudostratified columnar epithelium (neuroepitheliuma). These neuroepithelial cells, which were morphologically similar in any location, were undifferentiated and showed no sign of ependymal differentiation. There were frequent mitotic figures in the cells at the ventricular surface. At 18 days and even at 20 days of gestation, the ventricle was usually bordered by the neuroepithelium (Fig. la,b). One

Figure 1 Light micrographs of coronal sections through the (a.b) mid-brain of a control rat and (c.d) a hydrocephalic HTX rat at 20 days of gestation. In the hydrocephalic brain, the lateral ventricles are symmetrically dilated, and the neuroepithelium showed thinning with cellular disarrangement. Arrows in b and d indicate the width of the neuroepithelium.

1 18 Y. Nojirna eta/.

HTX rat had symmetrically dilated lateral ventricles. In this rat, the third ventricle was also slightly dilated, while the aqueduct was normal and open. Compared to the normal control rats (Fig.la.b), the HTX rat with dilated lateral ventricles showed thinning of the neuroepithelium, and cellular disarrangement associated with irregularly hyperchromatic nuclei and scant cytoplasm (Fig. 1c.d). Exudative macrophages were occasionally found within the ventricular lumen (Fig.2). However, the layer of neuroeplthelial cells was maintained. Spongiotic change in the subventricular zonem was not seen. No significant changes were found in the choroid plexus. No dilatation of the lateral ventricles was noted in three littermates of the hydrocephalic rat and in four rats from another litter, and their neuroepithelium had normal thickness with no cellular disarrangement.

At the age of 1 day after birth, four hydrocephalic HTX rats showed dilated lateral ventricles similar to the rats from 20 days of gestation, but the ventricular dilatation was stronger. The posterior horn of the lateral ventricles showed marked dilatation. The neuroepithelium at this site was stretched severely and the subventricular zone showed signifiiant spongiosis as shown by our previous study.’ The third ventricle and the aqueduct were also slightly dilated, compared to normal control rat brains. At 1 week after birth, ependymal differentiation in the lateral ventricles became clear in normal control rats. In hydrocephalic HTX rats,

Flgum 2 LgM micrograph of the lateral ventricular neuroepitheliurn in hydrocephalic HD( rat at 20 days of gestation. An aggregate of exudative macrophages (arrow) in the ventricular lumen is seen.

dilatation of the lateral ventricles and flattening of the ependyma showed further progression, while the aqueduct was narrowed and appeared to be occluded. The hydrocephalic HTX rats that survived more than 2 weeks after birth showed severe ventricular dilatation, associated with marked thinning of cerebral parenchyma. The ependyma of the dilated lateral ventricles showed the strongest thinning or had disappeared, and gliosis was found in the periventricular tissue.

lmmunohistochemical findings

Concerning the lateral ventricles, a small number of the neuroepithelial cells and none of the ependyma showed immunoreactivity for vimentin in normal control rats (Fig. 3a,c). In contrast, most of the neuroepithelium and ependyma facing the dilated lateral ventricles of HTX rats were positive for vimentin regradless of age (Fig. 3b,d). At 20 days of gestation, almost all neuroepithelial cells, including the cells showing disarrangement and nuclear deformity, were positive for vimentin (Fig.3b). However, in the third ventricle and the aqueduct, no significant difference in vimentin immunoreactivity of the neuroepithelium and ependyma was seen between hydrocephalic HTX rats and normal control rats.

Electron microscopic findings

Macroscopically, the manifestation of hydrocephalus was not detected in any of the HTX fetuses subjected to electron microscopic observation. Even in the ultrastructure, the neuroepitheliumm of HTX rat brains at 16 and 18 days of gestation did not show any differences from normal control rat brains. However, when compared to the age-matched normal control rats (Fig. 4a), the neuroepithelium of the lateral ventricles in the brains of HTX rats at 20 days of gestation showed cellular disarrangement (Fig. 4b). Some of them showed cellular deformity. The neuroepithelial changes, almost the same as those light-microscopically seen in the hydrocephalic 20-day-old fetal rat, were found in the dorsal and lateral walls of the anterior horn (Fig. 5a), as well as in the posterior horn (Fig.5b) of the lateral ventricles. These cells had an irregularly shaped nucleus with increasing numbers of small clumps of heterochromatin and dense nucleoplasm. In the scant cytoplasm there were numerous polyribosomes. Cytoplasmic blebs (Fig. 5a) were occasionally present. But cell organelles such as rough- surfaced endoplasmic reticulum, mitochondria, and a small Golgi complex were well preserved and there was no marked increase of intermediate filaments. On the other hand, the neuroepithelium of the third ventricle (Fig.5~) and the

Ependyma in congenital hydrocephalic rat 1 19

Figure 3 lmmunohistochemical staining for vimentin of the lateral ventricular neuroepithelium and ependyma in (as) normal rats and (b,d) hydrocephalic HTX rats at (a,b) 20 days of gestation and (c.d) 1 week after birth. Regardless of the prenatal and postnatal period, most of the neuroepithelium and ependyma of the hydrocephalic rats are strongly positive, whereas (a) a small number or (c) none of those of normal rats show immunoreactivity.

aqueduct (Fig.%) were almost similar in appearance to those in normal control rat brains. In the aqueduct the early differentiation of the ependyma was clearly identified by the formation of cilia (Fig.5d) and microvilli (Fig.5d) at the cell surface facing the ventricular lumen, although even the neuroepithelium in the lateral ventricles rarely showed a few cilia and microvilli. The number and distribution of the cilia did not differ from those in the lateral ventricles of normal control rat brains.

In the lateral ventricles of the 1 -day-old hydrocephalic HTX rats, neuroepithelial cells with dense, irregularly shaped nuclei and scant cytoplasm were more prevalent, particularly in the dorsal wall of the anterior horn and the posterior horn. They showed cellular disarrangement and widening of the intercellular spaces (Fig. 6a). The neuroepithelial cells at the ventricular surface maintained continuity with the neighboring neuroepithelial cells only by the inner-most intercellular junctional complexes. However, at the remaining sites on the lateral cell surfaces there was marked dissociation and microvilli were formed on the detached cell surfaces (Fig. 6b). There was an increase of macrophages infiltrating the neuroepithelium itself, and also into the subventricular zone.2o In contrast, the ependyma of the third ventricle and

aqueduct showed only slight disarrangement, without the significant changes observed in the lateral ventricles. In 1- week-old normal control rats, the ventricular system was almost entirely lined by a layer of cuboidal or columnar ependymal cells. In the hydrocephalic HTX rats, the intercellular spaces were widened further in the ependyma of the lateral ventricles, and the amounts of cilia and microvilli were decreased at the ventricular surface of the cell membranes, while the ependymal cells with dense and irregularly shaped nucleus and scant cytoplasm decreased in number and disappeared. In hydrocephalic HTX rats from 2 weeks after birth, stretching and flattening of the ependyma in the lateral ventricles had progressed further and the number of intermediate filaments in the cells was increased (Fig. 7). In the subependymal tissue, an axonal degeneration was observed. In a 4-week-old hydrocephalic HTX rat, dissoci- ation of the ependyma in the severely dilated lateral ventri- cles was reduced, while intermediate filaments (Fig. 8) of ependymal cells increased. Figure 9 shows the decreased number of cilia and microvilli in the flattened ependymal cells. Table 1 shows the summary of morphological findings in the lateral ventricle and lining of the neuroepithelium and ependyma of HTX rats at various ages.

120 Y. Nojima eta.

DISCUSSION

In the present study, the first onset of hydrocephalus in HTX rats was observed at gestational day 20 of gestation with dilatation of the lateral ventricles, which is associated with abnormalities of the neuroepithelium. At the Same gestational stage, also in one of the HTX rats without macroscopically visible ventricular dilatation, the neuroepithelial changes similar to those in the hydrocephalic fetal rat (is. cellular disarrangement and deformity in the neuroepithelium of the lateral ventricles) were observed by electron microscopy. The subsequent progression of hydrocephalus, as summarized in

Flgure 4 Electron micrographs of the lateral ventricular neuroepithelium in (a) a normal rat brain and (b) an HTX rat brain at 20 days of gestation. The HTX rat shows disarrangement and deformity of neuroepithelial cells with irregularly shaped, hyperchromatic nuclei and scant cytoplasm (arrows). L. ventricular lumen, V, blood vessel. Bar = 5pm.

Table 1, indicates that these neuroepithelial changes of lateral ventricles are the initial morphological event in the development of congenital hydrocephalus. It is suggested that these prenatal neuroepithelial abnormalities may be closely associated with the pathogenesis of this type of hydrocephalus. On the other hand, the neuroepithelial changes, even at a late gestational stage, may influence the migration of the neuroblasts and glioblasts into the mantle layer.

Recent studies on hydrocephalic models have also indicated that abnormalities of the ventricular ependyma may cause Nakamura and Sat0 reported that

Ependyma in congenital hydrocephalic rat 121

Figure 5 Electron micrographs of the neuroepithelium of the (a) anterior horn and (b) posterior horn of the lateral ventricle, (c) the third ventricle, and (d) the aqueduct from an HTX rat brain at 20 days of gestation. (a.b) In the lateral ventricle some neuroepithelial cells (arrows) show disarrangement and deformity. The neuroepitheliurn in the (c) third ventricle and (d) aqueduct appears not to be affected. In the latter, ependymal differentiation of the cells at the ventricular surface is seen. (a) Arrowhead, cytoplasmic bleb of neuroepithelial cell; (d) arrow, cilia; arrowhead, microvilli. Bars = 5pm.

122 Y. Nojima eta/.

ciliary movements were abnormal in the aqueductal ependyma of congenital hydrocephalic WIC-Hyd rats, and suggested that the disturbance of cerebrospinal fluid flow causes hydrocephalus.6 In the present study, however, no morphological abnormalities were apparent in the aqueductal neuroepithelium and ependyma of hydrocephalic HTX rats. Takano et a/. have reported that the inoculation of hamsters with mumps virus causes infection of the entire ependyma, with the lateral ventricles being particularly prone to functional disorders due to decreased numbers of cilia of ependymal cells, thus leading to dilatation of these ventricles.“ Our electron microscopic observations of the lateral ventricular neuroepithelium at 20 days of gestation revealed that cilia at the ventricular surface were few

Figure 6 (a) Electron micrographs of the lateral ventricular neuroepithelium of a 1-day-old hydrocephalic HTX rat, which shows increased cellular disarr- angement, deformity, and disassocia- tion. Bar = 5pm. (b) Widened intercel- lular spaces with microvilli along the lateral cell surface (arrows) and an increase in the amount of cytoplasmic microfilaments (arrowheads) are shown. Bar = 1 pm.

compared to the aqueduct. The distribution, however, was similar to that in the neuroepithelium of the lateral ventricles in control rats. Thus, a disturbance in the formation or movement of ependymal cilia in the lateral ventricles is unlikely to have caused hydrocephalus in HTX rats.

Electron microscopic evidence of an increase of ependymal intermediate filaments in hydrocephalic models have been reported previ~usly,’~~’’ but no systematic immunohistochemical observation has been done. In the present study, it was found that the imrnunostaining for vimentin was increased in the ventricular neuroepitheliurn and ependyrna of hydrocephalic rats.

Takano et a/. insisted that vimentin immunoreactivity is principally detected in immature ependymal cells and not in

Ependyma in congenital hydrocephalic rat 123

Figure 7 Electron micrograph of the ependyma in the lateral ventricle of a 2-weeks-old hydrocephalic HTX rat, which shows an increase in the amount of cytoplasmic intermediate filaments by about 10-11 nm in diameter (IF). Bar = 1wm.

Figure 8 (a) Electron micrographs of the ependyma and the subependyma of the lateral ventricle of a 4-weeks-old hydrocephalic HTX rat, which shows an increase in the amount of cytoplasmic intermediate filaments (arrowheads), axonal degeneration (arrows), and macrophage infiltration (M). Bar = 1 pm. (b) Higher magnification shows a marked increase in intermediate filaments. N, nucleus of ependymal cell. Bar = 0.5vm.

124 Y. Nojima eta/.

mature ependymal cells in normal human brains, whereas vimentin is expressed in immature ependymal cells in human hydrocephalic brains, both in prenatal and postnatal stages.23 The immature and mature ependymal cells in their report seem to be identical to the neuroepithelial and ependymal cells in the present study, respectively. Our results, therefore, are not contradictory to their results.

ACKNOWLEDGMENTS

The authors are grateful to Dr Y. Sada and Dr S. Kuwahara for their kind cooperation, and Mr T. Tokaji, Ms E. Miyazaki, Ms S. Yamasaki, Ms T. Matsui, Ms T. Kadota (First Department of Pathology, Kochi Medical School) and Mr M. Shirota (Medical Research Laboratory, Kochi Medical School) for their excellent technical assistance. The authors thank Professor K. Sat0 (Juntendo University School of Medicine, Tokyo, Japan) for kindly supplying the HTX rats used. This study was supported, in part, by a Grant-in-Aid for scientific research on ‘intractable hydrocephalus’ from the Ministry of Health and Welfare of Japan.

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Figure 9 Electron micrograph of the ependyma of the lateral ventricle of a 4- weeks-old hydrocephalic HTX rat showing the flattening of the cell and decreased number of cilia and microvilli at the ventricular surface (arrow). Bar = 3 pm.

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