experimental obstructive hydrocephalus in the rat: a scanning electron microscopic study

Neuropathology and Applied Neurobiology 1979, 5, 457468

EXPERIMENTAL OBSTRUCTIVE HYDROCEPHALUS I N

THE RAT: A SCANNING ELECTRON MICROSCOPIC STUDY

PATRICIA COLLINS Department of Anatomy, Downing Street, Cambridge CB2 3DY

Accepted for publication 21 February 1979

Collins P. (1975) Neuropathology and Applied Neurobiology 5,457468

Experimental obstructive hydrocephalus in the rat : a scanning electron microscopic study

Hydrocephalus was induced in 12-day old rats by the cisternal infusion of a concen- trated kaolin suspension. The animals were killed at day 20 and the ependyma1 lining of all the ventricles prepared for scanning electron microscopy. The dilation of the ventricles was moderate to gross in all cases. The ependyma of the lateral ventricles was similar in both control and experimental animals. Ependymal damage was present in six out of the twelve hydrocephalic rats. Two had fibres visible on the ependymal surface. Four had tears covered with small round cells, believed to be responsible for the repair of the ependyma. The third ventricle, cerebral aqueduct and fourth ventricle enlarged by incorporating folds of ependyma, present in control animals, into the ventricular walls. The circumventricular organs present in the third and fourth ventricles were not damaged by the dilation of the ventricles, even in severe hydrocephalus.

Introduction

Changes in the ependymal lining of the brain caused by hydrocephalus have been studied with light microscopy and with transmission electron microscopy by many workers (Russell, 1949; Weller & Wihiewski, 1969; Weller, Wihiewski, Ishii, Shulman & Terry, 1969; Torvik, Bhatia & Nyberg-Hansen, 1976). With the advent of scanning electron microscopy (SEM) the surface morphology of the ependyma can now be studied in greater detail. Using this technique nearly all of the ventricular lining of a hydrocephalic brain can be viewed and some measure made of the amount of ependymal damage caused by the condition. So far, SEM studies of hydrocephalus have been made by Nielsen & Gauger (1974) in the hamster, Page (1975) in the rabbit, and Go, Blaauw, Zuiderveen & Molenaar (1976) in the rat.

The main difference between the results of earlier studies of hydrocephalus and those more recently produced is in the observation of ependymal defects. Splitting of the ependymal layer has been reported in light microscopy studies (Weller & WiSniewski, 1969; Weller et al., 1969), but not in SEM studies (Page, 1975; Neilsen &

0305-1846/79/12oo-O457 $02.00 0 1979 Blackwell Scientific Publications 457

458 Patricia Collins

Gauger, 1974; Go et al., 1976). Further, no previous attention has been given to the appearance of the circumventricular organs in the hydrocephalic brain. These areas of specialized ependyma in the third and fourth ventricle are believed to be part of a system involved in the integration of blood, brain and cerebrospinal fluid (CSF) compartments (Scott, Sladek, Kozlowski, McNiel, Paul1 & Krobisch-Dudley, 1974). If these organs are indeed part of a vital system responsible for the neurotransmis- sion of information, then their alteration or damage by hydrocephalus may be of the greatest importance in its future management and treatment.

Materials and methods

Twenty-seven 12-day old Wistar-derived rate were used in this study. In twelve hydrocephalus was induced by the cisternal infusion of 0.8 ml of a thick suspension of kaolin in 0.9% NaC1. Six rats under- went the surgical procedure and were infused with 0.8 ml of 0.9% NaCl only. Six rats received the anaesthetic only (NembutalO.7 ml, 60 mg/ml) and three received no drugs or operative procedure.

At 20 days the animals were killed by aortic perfusion of Karnovsky’s fixative (pH 7.2 a t 4°C). The age of the rats ensures prepubescence, thus avoiding the oestrus cycle in the female which is known to alter the surface morphology of the infundibular ependyma. At this age the rats were still being suckled.

Each brain was dissected in the following way. The cerebellum and brain stem were separated from the cerebrum and midbrain. The roof and floor of the fourth ventricle were exposed. The fore and midbrain were divided longitudinally through the midline revealing the two lateral walls of the third ventricle. Slices were cut just lateral to the third ventricle opening up the lateral ventricles.

After dissection the specimens were placed in fresh fixative for 2 h then stored in 0.1~ sodium cacodylate buffer (pH 7.2) overnight at 4°C. Post-fixation was in 1% Os04 in 0 . 1 ~ sodium cacodylate buffer (pH 7.2) for 2 h, after which the specimens were dehydrated in increasing concentrations of acetone; the acetone was removed by the critical point method in a Polaron critical drying apparatus. The specimens were coated with gold in a Polaron E5000 sputter coater, then examined in a Cambridge S 600 stereoscan.

Results

After 8 days the rats which received kaolin had domed skulls, slight ataxia of the limbs and were smaller than their control litter mates. Examination of the brains showed kaolin in the subarachnoid spaces, around the brain stem, medulla oblongata and pons. Usually all four ventricles were dilated although the extent of the dilation varied from animal to animal.

Figures 1 & 2. Ependyma from the lateral ventricle of a control rat. 1. Shows moderately ciliated ependyma with microvilli. 2. Shows sparse cilia with fewer microvilli. This latter region was near a synechial area.

Figure 3. Ependyma from the lateral ventricle of a hydrocephalic rat. The cilia1 clumps are widely spaced and the microvilli on some cells are mainly around the cell borders. Three supra-ependymal macrophages can be seen.

SEM study of hydrocephalus in the rat 459


E X A M I N A T I O N O F T H E V E N T R I C U L A R W A L L S U S I N G S E M

Lateral ventricles

The general appearance of the lateral ventricle ependyma in the hydrocephalic animals was very similar to that observed in the controls (Figures 1-3). The cells had moderately spaced clumps of cilia and dense microvilli on the cell surface. In some of the experimental animals ‘bald’ cells were present, with no central clumps of cilia and the microvilli limited to the edge of the cells.

Two rats had fibres visible on top of, or through, the ependymal surfaces. In one animal, where the hydrocephalus was very severe, both lateral walls of the ventricles were without ependyma (Figure 4). In those areas still covered with ependyma the surface was similar to control except for the large numbers of supraependymal macrophages on the surface. The other example of fibres visible on top of the ependyma was not as dramatic. In the inferior angle of the ventricle the ependyma was non- ciliated with no apparent microvilli, parallel fibres were noted emerging then return- ing under the ependyma (Figure 5); a few round cells with processes were noted both on the ependymal surface and on the fibres.

Four experimental animals had tears in the lateral ventricle ependyma without visible fibres; in all cases these were on the lateral wall of the ventricle. The ependy- ma1 cells near the tears were ciliated and had microvilli, although ‘bald’ cells with microvilli confined to the border were also noted. The tears were covered with small

Figure 4. Lateral wall of the lateral ventricle in a severely hydrocephalic rat. The area E is covered with ependyma, area F is composed mainly of fibres covered with small round cells.


Figure 5. Ventricular lining from the inferior angle of the lateral ventricle of a hydrocephalic rat. This region is non-ciliated. Fibres are visible on the surface with small round cells on top of the fibres.

Figure 6. A tear in the ependymal surface of the lateral wall of the lateral ventricle of a hydrocephalic rat. The surrounding ependyma is sparsely ciliated; ‘bald’ cells are present. Small cells are covering the defect with their processes over both the defect and the ependymal cells.


round cells approximately 4-6 pm in diameter with long processes. These cells completely covered the defects but did not cover a larger area. The number of round cells over these tears were related to the size of the defect; some were covered by six to eight cells (Figure 6), others by more than sixty cells (Figure 7).

Third ventricle and cerebral aqueduct The ventricular ependyma was most densely and uniformly ciliated in the third ventricle and cerebral aqueduct. Here the cilia completely covered the ventricular surface and microvilli could not be observed. The direction of the cilia was con- sistently toward the aqueduct and fourth ventricle. Folds of ependyma were observed in the control animals around the massa intermedia and along the aqueduct.

In the hydrocephalic animals, although the third ventricle was notably expanded laterally and superiorly with the pineal recess being greatly enlarged (Figures 8 & 9),

Figure 9. Mid-sagittal section through the third ventricle of a hydrocephalic rat. The ependymal folds have been absorbed into the expanded ventricle. The distortion in shape caused by the hydrocephalus is mainly directed superiorly. The pineal recess is greatly enlarged.

Figure 7. Much larger tears in the lateral wall of the lateral ventricle of a hydrocephalic rat. This tear is covered by more than sixty cells.

Figure 8. Mid-sagittal section througn the third ventricle of a control rat. Folds of ependyma are present in the third ventricle and aqueduct. Massa intermedia (MI), choroid plexus (CP), pineal gland (PG), foramen of Monro (FM), pineal recess (PR), cerebral aqueduct (AQ) and fold (f) are visible.


Figure 10. Superior border of the massa intermedia in the third ventricle of a hydrocephalic rat showing the ependymal defect with fibres visible on the surface. Mama intermedia (MI), cerebral aqueduct (A&), foramen of Monro (FM) and tear (T) are visible.

the density of the cilia was not appreciably altered. However, the folds of ependyma around the massa intermedia and in the aqueduct were not present. In the majority of experimental animals the pineal recess was in continuation with the subarachnoid space. No change in the appearance of the ependyma over the circumventricular organs was observed. In three experimental animals tears were present in the ependyma on the superior border of the massa intermedia. Here the ependyma stopped abruptly and the surface of the tear was composed of fibres and small round cells 8-10 pm in diameter (Figure 10).

Fourth ventricle

In control animals the ciliated ependyma of the ventricle ranged from dense to moderately spaced. Only a marginal increase in the spacing of the cilia was noted in the hydrocephalic animals despite the large expansion of the ventricle. The most marked difference between control and experimental animals was in the presence and distri- bution of kaolin in the fourth ventricle (Figures 11 & 12). In the experimental animals, kaolin was evenly distributed over the brain stem and in the subarachnoid space, but that which had refluxed into the ventricle was in clumps. This clumping is attributed to the action of the supraependymal macrophages which were always in close association with the kaolin.


Figure 11. Floor of the fourth ventricle of a control rat. The walls are densely ciliated.

Figure 12. A similar view of the floor of the fourth ventricle of a hydrocephalic rat. Large clumps of kaolin are present on the ependyma. Clump of kaolin (C). Supraependymal macrophages are scattered over the surface.


Choroid plexus

The choroid plexus of all four ventricles were examined in all control and hydrocephalic animals. No alteration in the surface morphology was noted in the hydrocephalic animals.

Supraependymal macrophages

A marked macrophage response was noted in the kaolin treated animals: these results will be presented in another paper.

Discussion

The initial changes observed in the kaolin treated animals, i.e. ataxia, slower weight gain compared to controls, doming of the skull and distortion of the orbits have been reported in similar experimental animals by other workers (Weller et al., 1971; Torvik et al., 1976). The variation in the degree of hydrocephalus produced by the same quantity of kaolin was also noted by these workers. This is probably due to the development of spontaneous ventriculostomy in some animals.

Clark et al. (1970), suggested that new cells are added to the lateral ventricle ependyma in obstructive hydrocephalus. This concept was further promoted by Page (1975) who postulated that the new ependymal cells were those with microvilli confined to the borders of the cell and with no cilia. However, more recently this idea has been refuted by Weller et al. (1978). They counted the cells of the ependymal and subependymal layers in control and hydrocephalic rats and concluded that there is no significant increase in the number of ependymal cells present in the lateral ventricular walls in chronic hydrocephalus ; rather, the ependymal cells stretch to accommodate the enlargement of the ventricles.

In the present study moderate to severe enlargement of the ventricles was observed, but only relatively few cases of ependymal damage. However, in one animal with extreme ventricular dilation the destruction of the ependymal layer was gross. It may be that the ependyma will stretch only at a given rate, so that in animals with slowly developing hydrocephalus the cells can flatten and maintain their intercellular connections ; in animals with more acute onset and extreme ventricular dilation, the cells cannot stretch fast enough and ependymal tearing results.

Spaces between ependymal cells in hydrocephalic animals have been described in studies by Weller et al. (1969; 1971; 1978) and Price et al. (1976) using light microscopy and TEM; they were not observed in the scanning study of Go et al. (1976), while Torvik et al. (1976) in a light microscopic study interpreted such spaces as caused by rupture of the normal synechiae between the lateral ventricular walls. Weller et al. (1978) described ependymal defects in association with supraependymal macrophages and gliosis of the underlying brain tissue.


Tears in the ependyma were observed in six out of twelve hydrocephalic animals, in the present study; on occasion the destruction of the ependymal layer was marked. In the brains with moderage damage, small cells covered the defects. These cells had profiles differing from the supraependymal macrophages seen elsewhere in the ventricles in association with kaolin particles, being smaller and smoother with fewer processes and no ruffles. Similarly the cells did not fit the description of supraependymal neurones (Scott, Krobisch-Dudley, Paul1 & Koblowske, 1977).

It is suggested that these cells are specifically concerned with the repair of the ependymal layer. From the data obtained in this study it is not possible to say whether the tears would ultimately be repaired and the ependymal layer re-established, or whether a group of different cells would remain, i.e. a scar. The latter situation could result in an inelastic ventricular surface which would not respond fully to pressure fluctuations within the ventricle, thus possibly promoting more tears.

The enlargement of the ventricles caused by the hydrocephalus followed a pattern in all of the animals. Initially the lateral ventricles dilated, then the third and fourth ventricles. This order of events is contrary to the description by Russell (1949), but in agreement with Penfield & Elvide (1932). Further, the order of damage to the ependyma followed a similar pattern. Thus, the lateral ventricules enlarged stretch- ing the ependyma and ultimately producing tears. The third ventricle, cerebral aqueduct and fourth ventricle enlarged mainly by incorporating into their walls the folds of ependyma observed in control animals. Tears were observed on the border of the massa intermedia only in severe hydrocephalus.

Within the ventricles the distortion and damage to the ependyma was most often in areas covering white matter. These observations have been reported by Milhorat (1973) and Page (1975). To state this fact another way, the distortion and damage to the ependyma rarely occurred in areas covering grey matter.

In the third ventricle, cerebral aqueduct and fourth ventricle parts of the grey matter, particularly in the mid-line, are covered with morphologically distinct ependyma, collectively called the circumventricular organs. In the hydrocephalic rats it was noted that the circumventricular organs were not distorted or damaged by the ventricular dilation. Indeed in the severe cases the dilation of the lateral ventricles was extreme, but comparatively little enlargement of the third ventricle cerebral aqueduct and fourth ventricle was noted. This pattern of ventricular dilation produced significant damage to the lateral ventricle ependyma, little damage to the third ventricle ependyma and no damage to the circumventricular ependyma.

The conclusions I have made from this study are along two lines. First, gross ventricular enlargement sufficient to cause tearing of the ependyma may promote scar formation which could result in an inelastic ventricular surface which is even more prone to damage with exacerbation of the hydrocephalus. Secondly, the circumventricular organs are not damaged during the development of hydrocephalus, because of their situation, and because of the pattern of dilation of the ventricular system.

D


Acknowledgements

This study was completed while the author was in receipt of a Medical Research Council training grant. I wish to thank Professor R. J. Harrison for the provision of research facilities, Mr W. Moue1 for technical assistance and Mr J. F. Crane and his staff for photographic assistance.

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experimental obstructive hydrocephalus in the rat: a scanning electron microscopic study

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