Brain (1969) 92, 819-828. HISTOLOGICAL AND ULTRASTRUCTURAL CHANGES WITH Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 EXPERIMENTAL HYDROCEPHALUS IN ADULT RABBITS1 BY R. O. WELLER1 AND H. WISNIEWSKI (From the Department of Pathology (Newopathology) Albert Einstein College of Medicine, Bronx New York 10461) INTRODUCTION IN hydrocephalic infants with gross ventricular dilatation, the cerebral mantle is often extremely thin, especially over the convexities and the temporal poles. Despite this thinning, however, few histological accounts of brain tissue damage in hydrocephalus have been published. Russell (1949) emphasized the destruction of the ependyma around severely dilated ventricles and its replacement by subependymal glial cells. Struck and Hemmer (1964) found an increased extracellular space when they examined the cortex of hydrocephalic brains with the electron microscope; they also described alterations in cell organelles within neurones and glia. However, Friede (1962) observed no delay in the myelination of cerebral white matter in hydrocephalus. De (1950) described periventricular oedema and loss of ependyma in rats where hydrocephalus had been induced by the injection of indian ink into the cisterna magna. One disadvantage of De's method is that indian ink produces a marked inflammatory reaction and this may complicate the histological picture. The purpose of the present study is to investigate the histological damage in brain tissue caused by increased ventricular pressure alone. By using an inert, non-inflam- matory silicone oil to induce the hydrocephalus (Wi^niewski, Weller and Terry, 1969) the complications of ependymitis and leptomeningitis are avoided. The rabbit olfactory bulbs are used as models in this study for they present several advantages: First, they are remote from the site of the intracisternal injection of silicone oil; secondly, the olfactory bulbs are severely damaged in rabbit hydrocephalus whereas only minor tissue changes occur around the lateral ventricles. Thirdly, an xThis study was supported by Grant NB 02255 and NB 03356 from the National Institutes of Health. 'U.S. Public Health Service International Research Fellow. Fellowship Number 1 F05 TW 1263-01. Present address: Department of Pathology, Guy's Hospital Medical School, London, I S.E.I. 39 BRAIN—VOL. XCD 820 R. O. WELLER AND H. WiSNIEWSKI important feature of olfactory bulbs is the relative positions of white and grey matter; in this they resemble the convexities of the cerebral hemispheres with periventricular white matter and cortical grey matter. This last feature means that the histological changes in the rabbit olfactory bulbs may be correlated with tissue damage observed in human hydrocephalus. MATERIALS AND METHODS Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 Adult 3-4 kg. New Zealand white rabbits were used in the experiments; they were housed singly in wire cages and fed on rabbit bran. Hydrocephalus was induced by the subarachnoid infusion of inert silicone oils (Wis'niewski, Weller and Terry, 1969). One of two routes was used: either the direct injection of a very viscous oil (Dow-Corning 200 fluid, 100,000 centistokes) into the cisterna magna, or the indirect infusion of a lighter oil (Dow-Corning 200 fluid, 3,000 centistokes) through a polyethylene catheter in the spinal subarachnoid space. Seven animals were killed one to sixteen weeks after the infusion of the oil and four normal rabbits of similar ages were used for histological controls. Anaesthetized rabbits were killed by perfusion through the left ventricle of the heart with paraformaldehyde (100 ml.) followed by 5 per cent glutaraldehyde in 0-067 M phosphate buffer at pH 7-4 for fifteen minutes. Early in the perfusion the descending aorta was clamped, and a perfusion pressure of 80-100 mm. of mercury was maintained for fifteen minutes. The brain was quickly removed from the skull, and specimens were taken from the olfactory bulbs together with corpus callosum, centrum semiovale and cortex at the level of the anterior commissure. Coronal sections of the olfactory bulbs were made at the midpoint of the bulb, approximately 2-3 mm. from the distal end, and the whole coronal slice 0-5 to 0-75 mm. thick was post-fixed in osmium tetroxide (Dalton, 1955) for two and a half hours, dehydrated and embedded in Epon. 1 to 2 n thick Epon sections of the complete coronal face of the bulb were cut on a Reichert ultratome with glass knives and stained with 1 per cent toluidine blue. Areas were selected from these large sections and thin sections prepared, stained with uranyl acetate and lead citrate and viewed in a Siemens Elmiskop 1 electron microscope. The smaller pieces taken from the corpus callosum, centrum semiovale and cortex were treated in a similar manner for light and electron microscopy. Whole coronal sections of the cerebral hemispheres were dehydrated and embedded in paraffin, stained with haematoxylin and eosin, myelin stains and Holzer technique. RESULTS All the animals injected with silicone oil developed hydrocephalus but with some variation in the degree of dilatation of the lateral ventricles (fig. 1, Plate LX) and of the central lumina of the olfactory bulbs (fig. 2). Histological damage in the centrum semiovale and corpus callosum was slight. A few degenerating axons were found in these sites in severely hydrocephalic animals, but the ependyma remained intact. The olfactory bulbs, on the other hand, did suffer extensive tissue damage especially with the more severe hydrocephalus. Normal Olfactory Bulbs In the rabbit, the olfactory bulbs extend forward from the frontal lobes and are closely encased within sockets formed by the frontal and ethmoid bones. Each central ventricular lumen (figs. 2 and 3) is in direct continuity with the lateral I EXPERIMENTAL HYDROCEPHALUS IN RABBITS 821 ventricle; around the lumen are myelinated tracts formed by an extension of the anterior commissure and the olfactory tract (Ram6n y Cajal, 1955; Allison, 1953). Surrounding the white matter are strata of neurones and plexiform layers of non-myelinated processes (fig. 3). The innermost neurones are small granule cells and around their periphery is a ring of larger mitral cells (Ram6n y Cajal, 1955). This histologjcal arrangement is analogous to the distribution of white and grey matter in the convexities of the cerebral hemispheres. Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 Despite the adequate display of neuronal detail, paraffin embedded material was not satisfactory for study of the myeUnated layers. The myelin sheaths were, however, well visualized in 1 to 2 (i Epon sections stained with toluidine blue. Low power light microscopy showed two distinct layers of myelinated fibres in the normal rabbit olfactory bulb (fig. 4). The subependymal layer, with its loosely packed tissue elements, is encompassed by a denser layer of fibres (fig. 5). Electron- microscopically the loosely packed subependymal white matter shows an extensive extracellular space (Weller and Wisniewski, in preparation) which gradually diminishes to more normal dimensions in the more peripheral compact myelinated layers. Histological and Ultrastructural Changes in the Hydrocephalic Olfactory Bulbs Tissue damage in the olfactory bulbs increased with the greater degree of hydrocephalus. Two animals had mild histological changes, 4 animals showed severe acute tissue damage in the olfactory bulbs, whereas 1 rabbit with marked hydrocephalus had developed after sixteen weeks very severe degenerative changes in the white matter and complete loss of the ependymal lining. Changes in Mild Hydrocephalus The tissue changes in mild hydrocephalus were observed in the olfactory bulbs of 2 rabbits, 1 at eight days and the other at forty days following the subarachnoid infusion of silicone oil. In these animals, the lateral ventricles were only moderately dilated but the central lumina of the olfactory bulbs were increased to 2-5 times the normal cross sectional area (figs. 2 and 6). Histological examination at low power (fig. 6) showed an intact ventricular lining around the enlarged central lumen. One very noticeable change in the periventricular white matter, however, was the disappearance of the loosely packed subependymal layer; higher magnification (fig. 7) showed that this layer had become more compact. The neuronal layers, especially the granule cells, showed some increase in nuclear density and appeared to be compressed. Electron microscopic examination of the myelin in the compressed white matter showed no variation in the periodicity from the normal value of approximately 120 A. There was, however, a significant increase in the number of degenerating axons (fig. 8) in the compacted myeUnated tracts compared with the normal bulb where only very occasional fibres degenerate in young adult animals (Allison, 822 R. O. WELLER AND H. 1953). One of the degenerating fibres in fig. 8 shows the accumulation of mitochondria and dense bodies described by Webster (1962) in the early stages of wallerian degeneration in peripheral nerves. The electron microscopical studies also showed a reduction in the extracellular space in the compressed subependymal white matter. No ultrastructural abnormality was observed in the granule cells, mitral neurones, plexiform and glomerular layers. Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest