Histological and Ultrastructural Changes

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

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-inflammatory 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 on 29 September 2021 Severe Hydrocephalus Severe histologjcal changes were observed in the olfactory bulbs of four rabbits examined between five and forty days after the infusion of silicone oil. There was marked dilatation of the lateral ventricles in these animals; macroscopic examination of the olfactory bulbs revealed some dilatation of the central lumina and oedema of the periventricular tissues. Low power light microscopy of toluidine blue stained sections of the olfactory bulbs from each of these four animals confirmed the very marked oedema of the white matter; the apparently dilated central lumen proved to be a large paraventricular cyst (fig. 9). In some parts of the olfactory bulb the ependymal ring was still intact, whereas other sections showed continuity of the cyst with the ventricular lumen through a split in the ependymal lining (fig. 9). Even when the ependymal cells were separated from the underlying white matter they remained attached to each other and formed an intact layer. The paraventricular cysts were partly lined by flattened cells (fig. 10), possibly astrocytes, although some had cilia and may thus have been derived from ependyma. Electron microscopic examination of the free layer of ependyma showed that the luminal surface was normal and that the ependymal cell processes had formed a continuous pseudo- epithelial layer at the antiluminal surface (fig. 11) complete with tight junctions or terminal bar formation. Myelinated fibres were sparsely distributed throughout the cedematous white matter (fig. 10) indicating an extensive loss of nerve fibres. In addition, many axons were in the early stages of wallerian degeneration with the accumulation of dense bodies in the axons similar to fig. 8. The whole white matter was hypocellular with very little astrocytosis and almost no macrophages either around the vessels or between the widely separated myelinated fibres. Electron microscopy showed extensive extracellular oedema (fig. 12), and the absence of floccular material in the clear intercellular spaces suggests that the oedema fluid had a low protein content. Loose myelin profiles were found within the extracellular spaces and presented further evidence for the destruction of myelinated fibres. Many astrocyte processes containing bundles of coarse cytoplasmic fibrils were observed in the cedematous white matter. Furthermore, the astrocytes often contained cell and myelin debris within cytoplasmic vacuoles. Other cells had the characteristics of oligodendroglia; they sometimes had clear, smooth membrane-bound cytoplasmic vacuoles (fig. 12). Capillaries in the cedematous white matter appeared normal although in some cases there were gaps between the glial-basement membrane layer and the capillary pericyte (fig. 12). I EXPERIMENTAL HYDROCEPHALUS IN RABBITS 823

The outer neuronal layers in the cedematous olfactory bulbs appeared normal, but there was some cellular damage to the inner granule cell layers. Although the extracellular oedema did not spread between the cells of these layers there was swelling of astrocytes; and neurones (fig. 13) in the inner granule cell layers. One rabbit, which was killed sixteen weeks after the subarachnoid infusion of silicone oil, had developed extensive hydrocephalus with marked distension of the lateral ventricles (fig. 1). The central lumina of the olfactory bulbs in this case were Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 moderately dilated and the periventricular tissue was oedematous. Light microscopy showed complete destruction of the ependymal lining and only small groups of ependymal cells remained, usually clustered around blood vessels. There was extensive extracellular oedema of the white matter with the loss of large numbers of myelinated fibres. Many fibrous astrocyte processes were observed with the electron microscope in the oedematous white matter, but little overall increase in astrocytes was noted with the light microscope in H. and E. and Holzer preparations. Occasional macrophages were observed in the oedematous white matter situated mainly around blood vessels but very few were found within the tissue interstices. The outer grey matter was well preserved but some of the cells in the neuronal layers adjacent to the white matter were swollen and vacuolated.

DISCUSSION Hydrocephalus was produced in a total of 7 rabbits by the subarachnoid infusion of inert, non-infammatory silicone oils. During the development of the technique it was noticed that spontaneous ventriculostomy often occurred through a ruptured pineal recess; this tended to limit the degree of cerebral hydrocephalus obtainable in many of the rabbits (Wigniewski, Weller and Terry, 1969). This feature proved to be an advantage in the present study for a wide variation of hydrocephalus was thus obtained, and a range of tissue changes observed. Histological damage in the white matter and cortex surrounding the lateral ventricles was difficult to detect. The olfactory bulb, on the other hand, has proved to be a useful model for the study of tissue changes that result from increased intraventricular pressure. With increasing severity of hydrocephalus a sequence of tissue changes was seen in the olfactory bulbs. Mild ventricular dilatation caused some compression of the white and grey matter resulting in the elimination of the subependymal extracellular space. Very little tissue damage was observed at this stage and the ependyma remained intact. More severe hydrocephalus, however, caused splitting of the ependyma and extensive extracellular oedema of the white matter with destruction of its component myelinated fibres. The extracellular fluid was very clear, and appeared to contain little protein; this suggests that it may be cerebrospinal fluid from the ventricle, forced into the white matter through the tear in the ependyma rather than exudate from the blood. Despite the extensive destruction of the white matter, little damage was detected in the grey matter except I for some vacuolation and swelling of the astrocytes and neurones immediately 824 R. O. WELLER AND H. WlSNIEWSKI adjacent to the white matter. There was, however, no extracellular oedema in the neuronal layers. The difference in distribution of oedema fluid in white and grey matter in the present case is similar to the pattern of inflammatory oedema, but the origin of the fluid is probably different. White matter oedema induced by the implantation of cryptococcal polysaccharide pellets (Hirano, Zimmerman and Levine, 1965) or by tumour implants (Herzog, Levy and Scheinberg, 1965) is mainly extracellular,Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 whereas oedema fluid in the cortex accumulates within the cells (Torack, Terry and Zimmerman, 1959). Some of the features in the pattern of histological damage seen in the present study have been described in both human and experimental hydrocephalus by previous authors. Widespread loss of the ependymal lining of the lateral ventricles has been described in hydrocephalic human infants (Russell, 1949) and in adult hydrocephalic rats (De, 1950). In both these instances, however, the complication of ependymitis could not be ruled out. De (1950) also found severe peri ventricular oedema of both white and grey matter in his adult hydrocephalic rats. Fishman and Greer (1963) found an increase in the water, sodium and chloride content of the white matter in hydrocephalic adult dogs, but a decrease in protein, lipid and potassium. These findings are consistent with an increase in extracellular fluid but, again, their results must be interpreted with caution as they injected kaolin into the ventricles to produce the hydrocephalus and this causes a gross inflammatory reaction (Schurr et ah, 1953). The present investigation has emphasized that the white matter may be severely damaged when the intraventricular pressure is raised, whereas the grey matter may remain almost completely intact. Penfield and Elvidge (1932) suggested that the vascular supply of the white matter may be obliterated first during a rise in intraventricular pressure. This might account for the early damage to the white matter, but neither De (1950) nor Hassler (1964) found conclusive evidence of damage to blood vessels in the white matter of either human or rat brains with hydrocephalus. An important feature described in the present study is the difference in extent of the "cerebrospinal fluid oedema" in white and grey matter. Whereas the oedema fluid spreads extensively throughout the extracellular space of the white matter, it does not involve the grey matter; possibly the cerebrospinal fluid oedema is a majdr cause of the white matter destruction. The adult animal cannot be used as an exact analogue to study the events occurring in human infant hydrocephalic brains, as these are usually poorly myelinated and are enclosed by a more distensible skull; nevertheless, from the present study a working hypothesis may be tentatively suggested. White matter damage that occurs in hydrocephalus as a result of compression may be enhanced by the cerebrospinal fluid oedema that follows disruption of the ependyma. The slow destruction of the white matter in this manner may lead to the extreme thinning of the cerebral mantle. Further support for this hypothesis will be forthcoming from the studies of hydrocephalic infant human and puppy brains that are in progress. I EXPERIMENTAL HYDROCEPHALUS IN RABBITS 825

SUMMARY Hydrocephalus was induced in adult rabbits by the infusion of inert silicone oils into the cisterna magna and spinal subarachnoid space. The olfactory bulbs were the most severely affected regions of the brain; from the study of toluidine blue stained 1 to 2 \i Epon sections and electron micrographs, a possible sequence of tissue damage in hydrocephalus has been proposed. In mild hydrocephalus the central lumen of the bulb was dilated and the ependyma was stretched; in addition, Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 the neuronal layers and portions of the white matter were compressed. More severe hydrocephalus was accompanied by splitting of the ependyma, gross extracellular oedema of the white matter and destruction of nerve fibres. After sixteen weeks of progressive hydrocephalus the white matter was spongy, atrophic and oedematous. Very little damage occurred in the neuronal layers, but even this was confined to astrocyte and neuronal swelling in the deeper granule cell layers; the outer neurones were well preserved throughout. It is proposed that the splitting of the ependymal lining and the ensuing free flow of C.S.F. into the white matter may enhance any tissue damage caused by the compressive effects of hydrocephalus. This slow destruction of brain tissue may lead to the extreme thinning of the cerebral mantle seen in hydrocephalus.

ACKNOWLEDGMENTS We would like to thank Dr. Robert Terry for his advice and criticism, and Mr. Milton Kurtz for the photography of the gross brain specimens.

REFERENCES ALLISON, A. C. (1953) The structure of the olfactory bulb and its relationship to the olfactory pathways in the rabbit and the rat, J. comp. Neurol., 98, 309-350. DALTON, A. 5. (1955) A chrome-osmium fixative for electron microscopy, Anal. Rec, 121, 281. DE, S. N. (1950) A study of the changes in the brain in experimental internal hydrocephalus, /. Path. Bad., 62,197-208. FISHMAN, R. A., and GREER, M. (1963) Experimental obstructive hydrocephalus. Changes in the cerebrum, Archs Neurol. Chicago, 8, 156—161. FRIEDE, R. L. (1962) A quantitative study of myelination in hydrocephalus (factors controlling glial prolifera- tion in myelination), /. Neuropath, exp. Neurol., 21, 645-648. HASSLER, O. (1964) Angioarchitecture in hydrocephalus. An autopsy and experimental study with the aid of microangiography, Ada neuropath., 4, 65-74. HERZOO, I., LEVY, W. A., and Scheinberg, L. C. (1965) Biochemical and morphologic studies of cerebral oedema associated with intracerebral tumors in rabbits, /. Neuropath, exp. Neurol., 24, 244-255. HIRANO, A., ZIMMERMAN, H. M., and LEVTNE, S. (1965) Fine structure of cerebral fluid accumulation. VI. Intracellular accumulation of fluid and cryptococcal porysaccharide in oligodendroglia, Arclis Neurol, Chicago, 12, 189-196. PENFIELD, W., and ELVIDGE, A. R. (1932) Hydrocephalus and the atrophy of cerebral compression. In: "Cytology and Cellular Pathology of the Nervous System." Edited by W. Penfield. N.Y.: Hoeber. I Vol. 3, pp. 1203-1217. 826 R. O. WELLER AND H.

RAM6N Y CAJAL, S. (1955) Histologie du systeme nerveux de rhomme et de vertibres. Madrid: Instituto Ram6n y Cajal. Vol. 2, pp. 647-674. RUSSELL, D. S. (1949) Observations on the pathology of hydrocephalus. Spec. Rep. Ser. med. Res. Coun., No. 265, 119-122. SCHURR, P. H., MCLAURIN, R. L., and INGRAHAM, F. D. (1953) Experimental studies on the circulation of the cerebrospinal fluid and methods of producing communicating hydrocephalus in the dog, /. Neurosurg., 10, 515-525.

STRUCK, G., and HEMMER, R. (1964) Elektronenmikroskopische Untersuchungen an der menschlichenDownloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 Hirnrinde beim Hydrocephalus, Arch. Psychiat. NervKrank., 206,17-27. TORACK, R. M., TERRY, R. D., and ZIMMERMAN, H. M. (1959) The fine structure of cerebral fluid accumulation. I. Swelling secondary to cold injury, Am. J. Path., 35, 1135-1140. WEBSTER, H. DEF. (1962) Transient, focal accumulation of axonal mitochondria during the early stages of wallerian degeneration, /. Cell. Biol., 12, 361-377.

WELLER, R. O., and WIJ'NIEWSKI, H. In preparation. WISMEWSKI, H., WELLER, R. O., and TERRY, R. D. (1969) Experimental hydrocephalus produced by the subarachnoid infusion of silicone oil, /. Neurosurg., 31,10-14.

I EXPERIMENTAL HYDROCEPHALUS IN RABBITS 827

LEGENDS FOR PLATES PLATE LX Fio. 1.—Coronal section of adult rabbit brain showing hydrocephalus of the lateral ventricles sixteen weeks after the infusion of silicone oil into the cisterna magna via the spinal subarachnoid space.

FIG. 2.—Coronal sections through the olfactory bulbs of normal (above) and mildly hydro- Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 cephalk (below) rabbit (forty days after infusion of oil). In the hydrocephalic animal, the central ventricular lumina are dilated. FIG. 3.—Haematoxylin and cosin stained coronal section of normal olfactory bulb. 1, Central ventricular lumen lined by ependyma. 2, White matter including anterior commissure and olfactory tract. 3, Granule cell layer. 4, Inner plexiform layer. 5, Mitral cell layer. 6, Outer plexiform layer. 7, Glomerular layer (Ramon y Cajal, 1955). x 38.

PLATE LXI FIG. 4.—Normal olfactory bulb showing the lightly stained subependymal layer (a) and the more heavily stained outer layer of myelinated fibres (b). Toluidine blue stained Epon section. X40. FIG. 5.—Higher power view of normal olfactory bulb showing a loosely packed layer of myelinated fibres in the subependymal region (a), and an outer compact layer of myelinated fibres. Ependyma (e). Toluidine blue stained Epon section, x 400. FIG. 6.—Mild hydrocephalus. Low power view showing dilated ventricular lumen of the olfactory bulb. The loosely packed subependymal myelinated layer is no longer discernible. Toluidine blue stained Epon section. x40. FIG. 7.—Higher power view of fig. 6 to show compaction of the subependymal layer of myelinated fibres (compare fig. 5). Ependyma (e). Toluidine blue stained Epon section. X400.

PLATE LXU FIG. 8.—Electron micrograph of the compressed myelinated layers in a mildly hydrocephalic rabbit showing two degenerating fibres. The myelinaxed fibre in the centre has a swollen axon which contains numerous mitochondria and dense bodies; another degenerating axon is dark and granular (g). Uranyl acetate and lead stained, x 11,500. FIG. 9.—Severe hydrocephalus of olfactory bulb showing extremely cedematous white matter. Through a split in the ependymal lining there is continuity of a paraventricular cyst with the ventricular lumen. The ependyma has peeled away from the underlying white matter (v). Toluidine blue stained Epon section, x 100. FIG. 10.—Light micrograph showing flattened cells (f), possibly astrocytes, lining the paraventricular cyst, in a severely hydrocephalic olfactory bulb. The underlying white matter is grossly cedematous. Ependymal cells (e) still form an intact sheet seen at higher magnification in fig. 11. Toluidine blue stained Epon section. x250. FIG. 11.—Electron micrograph of dissociated ependymal lining showing intact cell layer with normal microvilli and cilia on the luminal surface (L). Flattened ependymal processes on the antiluminal border (AL) form a continuous layer with terminal bars between them. Uranyl acetate I and lead citrate stained, x 2,100. 828 R. O. WELLER AND H. WI^NIEWSKI

PLATE LXm FIG. 12.—Electron micrograph of cedematous white matter in severely hydrocephalic olfactory bulb. There are a few normal fibres around the capillary (cap) but there are also myelin profiles (m), probably from degenerating fibres. The intercellular spaces are very clear. Some separation of glial processes (gl) from the capillary pericytes is also observed (*) and some vacuolation of glial cells (vac). Capillary endothelium (en). Uranyl acetate and lead citrate stained, x 3,500.

FIG. 13.—Electron micrograph showing swollen vacuolated cells that are probably neuronesDownloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021 (N) within the granule cell layer adjacent to the oedematous white matter in a severely hydrocephalic olfactory bulb. There is no increase in the extracellular space. Stained with uranyl acetate and lead citrate, x 3,500.

{Received 5 February 1969)

I PLATE LX Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021

I To illustrate article by R. O. Weller and H. Wi'sniewski. PLATE LXI Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021

To illustrate article by R. O. Weller and H. Wisniewski. I PLATE LXII Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021

I AL To illustrate article by R. O. Welter and H. Wisniewski. PLATE LXI1I Downloaded from https://academic.oup.com/brain/article/92/4/819/419343 by guest on 29 September 2021

I To illustrate article by R. O. Weller and H. Wisniewski.