Infantile type of so-called neuronal ceroid-lipofuscinosis

Journal of the neurological Sciences, 18 ( 1973) 269-285

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Infantile Type of so-called Neuronal Ceroid-Lipofuscinosis Part 2. Morphological and Biochemical Studies M. HALTIA, J. RAPOLA, P. SANTAVUORI AND A. KERANEN The Children's Hospital, 2nd Department of Pathology, and Department of Medical Chemistry, University of Helsinki, Helsinki (Finland) (Received 18 August, 1972)

INTRODUCTION

Since the original description of late infantile amaurotic idiocy by Jfinsk2~(1913) and Bielschowsky (1913), a limited number of papers concerning the histological, histochemical, ultrastructural and biochemical features of similar cases has appeared, recently reviewed by Zeman, Donahue, Dyken and Green (1970). Like other types of so-called neuronal ceroid-lipofuscinoses (NCL) (Zeman and Dyken 1969) the Jfinsk2~Bielschowsky type of amaurotic idiocy is characterized by the accumulation in nerve cells of yellowish autofluorescent granules with the tinctorial properties of ceroid or lipofuscin. Additional histological features include a severe cerebellar atrophy, already stressed by Bielschowsky, and the occurrence of spheroid inclusions, particularly in the neurons of the thalamus and substantia nigra (Seitelberger 1962; Seitelberger, Jacob and Schnabel 1967). The neuroglial and microglial reactions are said to be insignificant. Biochemical studies have not revealed any consistent changes, and particularly no specific alterations of ganglioside metabolism (Zeman et al. 1970). The results of recent ultrastructural studies appear to fall into two separate categories. While older patients with NCL have shown a variety of abnormal cellular deposits, most reports on patients whose disease began before the age of 4 years have shown the almost exclusive presence of groups of laminated structures in the neuronal cytoplasm, variously designated as multilocular bodies (Zeman and Donahue 1963), multilamellar cytosomes (Gonatas, Gambetti and Baird 1968), aggregates of smooth membranes (Richardson and Bornhofen 1968), curvilinear bodies (Duffy, Kornfeld and Suzuki 1968), and cytosomes with curvilinear profiles (Carpenter, Karpati and Andermann 1972). We have recently been able to examine brain biopsy material morphologically and biochemically from 12 patients with a disease resembling late infantile amaurotic This work was supported by the Emil Aaltonen Foundation and the National Research Council for Medical Sciences, Finland.

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idiocy which clinically differ from most cases published previously, particularly by the early onset of the disease at around 1 year of age, absence or relative paucity of convulsions, and early amaur osis (Santavuori, Haltia, Rapola and Rait ta 1973). Hist ologically these cases, as well as an additional autopsy case, were characterized by severe neuronal destruction accompanied by massive occurrence of frequently multinucleated phagocytic cells. Both neurons and glial cells contained granular material which histochemically resembled lipofuscin but ultrastructurally differed both from ordinary neuronal lipofuscin and from the storage material described in most previous reports of late infantile amaurotic idiocy. PATIENTS AND METHODS

Patients: The series consists of 13 patients, boys and girls, clinically examined at the University of Helsinki Children's Hospital (Santavuori et al. 1973). From 12 patients brain biopsy material was obtained for morphological and biochemical investigation. From 1 additional patient (Case 11) only formalin-fixed autopsy material from the cerebral cortex, cerebellum and brain stem nuclei was available. Brain biopsy technique: After craniotomy a piece measuring approximately i cm 3 in volume and including both cortex and subcortical white matter was taken from the right medial frontal gyrus under general anaesthesia. Immediately after removal the tissue was divided with a razor blade into three samples for histological, electron microscopic and biochemical studies. Histological technique: The material was fixed in 10 ~ neutral formalin and a part of it was embedded in paraffin. The following stains were applied on paraffin sections: haematoxylin-eosin, van Gieson, luxol fast blue-cresyl violet, Palmgren's or Holmes' silver impregnation, periodic acid-Schiff (PAS) method according to McManus, Sudan black B, Long Ziehl-Neelsen. Frozen sections were treated in the following ways : Cajal's gold chloride-sublimate method for astrocytes, PAS (McManus), Sudan black B, Scarlet red, Oil red O, osmium tetroxide-beta-naphtylamine (OTAN) (Adams 1965), Holczinger's copper rubeanic acid method for free fatty acids (Adams 1965). In addition, both paraffin sections and frozen sections mounted in glycerin were examined in ultraviolet light using a Leitz Panphot microscope equipped with an Osram HBO 200 mercury lamp and a Schott UG1 (4 mm) energy filter and a Leitz K 430 barrier filter. Fresh frozen sections from Case 9 were fixed in 10 ~ formalin containing 2 ~oCaC12, pH 7.0, for 10 min, and assayed for acid phosphatase activity (Lake 1966). In Cases 12-15 the acid phosphatase activity was also estimated by the method of Barka and Anderson (1963) using naphthol AS-TR-phosphate as substrate. Electron microscopic technique : Approximately 1 mm 3 pieces of the cortical gray and white matter were fixed in chilled 2 ~o glutaraldehyde solution buffered to pH 7.2 with 0.1 M cacodylate buffer. After 2 to 6 hr fixation the pieces were rinsed in several changes of the same buffer overnight or over several days and post-fLxed in phosphatebuffered (pH 7.2) 1 ~ osmium tetroxide for 90 min. After dehydration in graded alcohols the tissue was embedded in Epon 812. Toluidine blue-stained thick sections (1

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#m), cut with a Porter Blum (Sorvall) microtome, were used for orientation. Thin sections, laid on uncoated copper grids, were stained with lead citrate (Venable and Coggeshall 1965) or double-stained with uranyl acetate and lead citrate and studied at 60 kV in an electron microscope (Zeiss EM-9A). Biochemical methods: In 9 cases (Cases 2, 3, 5, 6, 7, 9, 11, 12 and 13) the brain samples intended for biochemical analysis were stored in chloroform-methanol 2:1 (v/v) at 20°C until extraction. Isolation of gangliosides was performed according to FolchPi. Lees and Sloane-Stanley (1957). The tissue samples were homogenized with chloroform-methanol 2:1 (v/v). The total lipid extract was washed once with 0.1 ~o KC1 solution and twice with "theoretical upper phase" (chloroform-methanol-0.1 ~o KC1 3 : 48 : 47, all by vol). The resulting upper phases were combined and dialyzed against running tap water for 24 hr. The dialysate was dried and the dry rest was dissolved in chloroform-methanol 2 : 1 (v/v). The "crude ganglioside fraction" obtained was further purified by silicic acid column chromatography essentially according to McCluer, Coram and Lee (1962). The ganglioside fraction obtained from the silicic acid column was analysed on thin layer chromatography (TLC) using plates coated with Silicagel G (Merck, Darmstadt). Elution solvents described by Penick, Meisler and McCluer (1966)were used. Standards used were kindly proposed by Prof. E. Klenk (GM 1, GDla, GDIb, GT1), Dr. J. Palo (GM2) and Dr. K. Puro (GM a, both N-acetyl- and N-glycolyl-neuraminic acid type). The nomenclature described by Svennerholm (1964) is used throughout the text. Lipid-bound N-acetyl-neuraminic acid (NANA) was estimated according to Miettinen and Takki-Luukkainen (1959). -

RESULTS

Histological observations The histological picture in all 13 cases was qualitatively stereotyped and only the degree of the tissue alterations varied (see Table 1). All the cases will therefore be treated together. Changes in the frontal cerebral cortex. In all cases the cerebral cortex appeared very thin. Even in the youngest patients the cortical cytoarchitecture was severely disturbed. There was a marked loss of neurons. The remaining nerve cells had large vesicular nuclei and their scanty cytoplasm was distended by a granular material (Figs. 1A, 2A). In some neuronal perikarya larger roundish or irregular homogeneous cytoplasmic inclusions were present with staining characteristics similar to those of the granular material. The inclusions were often surrounded by a vacuole (Figs. 2B, C). The Nissl substance and neurofibrils were scanty. Between the cortical neurones great numbers of phagocytes were seen. These cells contained coarse granules with somewhat different staining characteristics. The nuclei of the phagocytes were small, round and hyperchromatic and some phagocytes had two or even three nuclei (Figs. 1C, 6C). Cuffs of these cells were seen around some blood vessels. Neuronophagia was not a prominent feature. There was an intense cortical fibrillary astrogliosis (Fig. 3A). The hypertrophic astrocytes had very coarse processes and contained granular storage material similar to that of the nerve cells.

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Fig. 1. A: A relatively well-preserved area of the cerebral cortex from a boy aged 1 year 8 months (Case 3). Note the accumulation of PAS-positive material within the scanty cytoplasm of cortical neurons and in a number of phagocytic cells. Paraffin, PAS (McManus), × 790. B: The same case. Autofluorescent granules within nerve cell bodies (semilunar structures with weaker autofluorescence) and phagocytes (more intense autofluorescence). Unstained paraffin section, ultraviolet light, x 790. C: Cerebral cortex from a boy aged 4 years (Case 5). Note the disappearance of neurons and massive occurrence of phagocytes containing PAS-positive material. Paraffin, PAS (McManus), x 790. D : Cerebral cortex and subcortical white matter (lower left corner) from the same case. The thin cortical ribbon is entirely devoid of nerve cells and harbors great amounts of strongly autofluoreseent phagocytes. Between them hazy, less intensely autofluorescent astrocytes are visible. Unstained paraffin section, ultraviolet light, x 180.

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Fig. 2. A : Granular storage material within the cytoplasm of a cortical neuron ; B : A n irregular conglomeration of coarse storage granules surrounded by a halo; C: A roundish "inclusion body" surrounded by ~ halo in the cytoplasm of a small cortical neuron.

In the older patients there was a subtotal to total loss of nerve cells from the cerebral cortex. The only cells left were characteristic phagocytes in great numbers seen in a feltwork of hypertrophic fibrillary astroglia and capillaries (Fig. 1C). In some places the tissue attained a spongiform character. The leptomeninges were thick and contained scattered mononuclear cells. Changes in the subcortical white matter. In the youngest patients the white matter appeared to be relatively spared. Most of the myelin sheaths and axons appeared to be preserved. There was, however, a considerable increase in the number of astrocytes and these appeared hypertrophic and contained granules with staining characteristics similar to those of the nerve cells. Only very few macrophages were seen, mainly around some blood vessels. In the older patients there was a subtotal to total loss of axons and myelin sheaths with accompanying pronounced fibrillary astrogliosis (Fig. 3D). In these cases the astrocytes contained considerable amounts of the granular material, and scattered macrophages were seen diffusely in the white substance and around the blood vessels. Changes in other parts of the brain : Changes in other parts of the cerebral cortex, basal ganglia, brain stem and cerebellum could only be studied in the autopsy case (Case 11). All samples from the cerebral cortex showed a total loss of neurons and a spongy network of fibrillary astroglia and capillaries harboring varying numbers of phagocytes. In the basal ganglia and brain stem nerve cells could still be seen. All of them were swollen, their cytoplasm containing great amounts of the characteristic granular storage material. In addition, a large number of macrophages were seen. In the cerebellum all the Purkinje cells as well as the granular cells had disappeared. Instead, there was a thick rim of hypertrophic Bergmann's glia with scattered macrophages (Fig. 3C) and the hypertrophic glial cells contained considerable amounts of the storage material. In all parts of the brain there was a subtotal to total loss of myelin with accompanying fibrillary astrogliosis. H istochemical observations The histochemical reactions of the stored substance were similar in all cases. The

Fig. 3. A : Cerebral cortex showing hypertrophic astrocytes with coarse processes (Case 3). Cajal, × 350. B : Astrocytes and phagocytes from the cerebral cortex showing intense acid phosphatase activity (Case 9). Lake s method, × 790. C: Cerebellar folium from case 11. Note the total disappearance of Purkinje cells and granule cells. A rim of hypertrophic astrocytes and a few phagocytes indicate their previous site. Paraffin, PAS (McManus), × 180. D: Subcortical white matter showing subtotal myelin loss; two fragments of myelin sheaths are still visible. Note the increase in the number of glial cells (Case 5). Paraffin, luxol fast blue-cresyl violet, × 790.

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material contained in the nerve cells, phagocytes and astrocytes was very resistant to various lipid solvents including chloroform-methanol (2 : 1). In unstained sections it was colourless or slightly yellowish. It was autofluorescent in ultraviolet light (Figs. 1B, D). The granules were acid-fast (long Ziehl-Neelsen), strongly PAS-positive (Figs. 1A, C, 2A) and stained intensely black with Sudan black B. Both with Scarlet red and Oil red O the material stained orange-red. The material was stained black by the O T A N method and it seemed to be stained by Holczinger's copper-rubeanic acid method for free fatty acids.

Fig. 4. Neuronal cytoplasm with storage material mixed with the rough-surfaced endoplasmic reticulum. There are some small spherical globules and the larger accumulations (arrows) seem to be formed by fusion of the small units, × 20,000.

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There were, however, some differences between the granular material contained in the nerve cells and the phagocytes. While the granules of the nerve cells gave a yellow to orange autofluorescence in ultraviolet light, the material in the macrophages showed a more intense greenish colour (Fig. 1B). The neuronal storage substance stained deep blue with luxol fast blue but the macrophages showed strong basophilia staining heavily with cresyl violet. Strong acid phosphatase activity was demonstrated in the storage granules in the cases assayed for this enzyme (Fig. 3B).

Electron microscopy The ultrastructure was examined in all 12 biopsy cases. The findings were uniform in all cases, except that the neurons were extremely difficult to identify in part of the material. Apart from the paucity of neurons in older patients, the age of a patient or the time from the commencement of the symptoms did not affect the electron-microscopic picture. As would be expected from the findings in the light microscopy, the most prominent feature was the presence of the electron-dense storage material in the cytoplasm of several cell types. Macrophages, astrocytes, and occasionally endothelial and perithelial cells contained the storage material as well as the neurons. The deposits often occupied about half of the perikaryonal area of the neurons and were mixed between the vesicles of the rough-surfaced endoplasmic reticulum (Fig. 4). There was no close association of the endoplasmic reticulum or any other organelles

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Fig. 5. A : Higher magnification of the storage material. The globule in the lower left corner shows concentric lamination. In the upper part of the picture part of a larger storage accumulation with a limiting membrane (arrows) is seen. This storage material is granular without any special structures, x 76,000. B: On the left is part of a myelinated (M) axon containing granular storage material (S). On the right, processes of hypertrophic fibrillary astrocytes (F) are seen, x 48,000.

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with t h e s t o r a g e m a t e r i a l . T h e d e p o s i t s v a r i e d in shape a n d size, b u t the basic u n i l s e e m e d to be a r o u n d or oval i n c l u s i o n 0 . 1 - 0 . 5 / ~ m in d i a m e t e r (Figs. 4 a n d 5A). The s t r u c t u r e of m a n y larger d e p o s i t s suggested t h a t they were f o r m e d by fusion of the

Fig. 6. A : A hypertrophic astrocyte with thick bundles of fibrils (F) in the white matter, x 5,000. B : A collection of spherical storage globules surrounded by glial fibrils (arrows), x 20,000. C : A binucleated macrophage, the cytoplasm of which is filled with the electron-dense storage material, x 5,000.

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small spherical globules (Fig. 4). Similar deposits were often seen in axons (Fig. 5B). On higher magnification concentric laminar structures were occasionally seen in the small deposits (Fig. 5A). The thickness of the dense lines in these laminations was approximately 85 A. Some remnants of the linear structures were occasionally detected in the larger deposits, but their matrix was mainly granular with varying electron density and without any detectable structures (Fig. 5A). The deposits were at least partially surrounded by a limiting membrane (Fig. 5A). Very rarely an ovoid electronopaque lipid globule was associated with these deposits• No cytosomes with curvilinear profiles nor any storage material with a typical finger-print pattern were detected. Another prominent feature was the abundance of hypertrophic fibrillary astrocytes as shown by light microscopy. The bodies of the astrocytes sent out very thick fibrillary processes (Fig. 6A) and these processes were seen all over in almost every field of view in both gray and white matter (Fig. 5B). The storage deposits in the astrocytes were less prominent than in the neurons and they usually showed up as clusters of the small globules (Fig. 6B). The macrophages were the most numerous cell type in almost all cases. Even in the electron micrographs several binucleated cells were often seen (Fig. 6C). The cytoplasm of the macrophages was filled with the electron-dense granular storage material.

Biochemicalfindings The results of the lipid-bound NANA analyses are expressed in Table 2. It is apparent that there was a marked fall in the concentration of lipid-bound NANA in all cases examined. The reduction was most pronounced in the oldest patients. In the thin-layer chromatograms the ganglioside pattern in the youngest patient (Case 3) seemed to be normal. In all other cases there seemed to be slight changes in the ganglioside pattern. In Cases 2, 5, 6 and 11 there was a rise in the relative amount of N-acetyl-neuraminyl-lactosylceramide (GM3), which is present only in minute amounts in the normal brain. Although there appeared to be some quantitative chanTABLE 2 C O N T E N T OF L I P I D - B O U N D N - A C E T Y L N E U R A M I N I C

ACID

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IN T H E C E R E B R A L C O R T E X F R O M P A T I E N T S

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Case number

Age (years)

N A N A content (#a/ma of wet tissue)

2 3 5 6 7 9 11 12 13 Normal controls*

2.4 1.7 4.0 2.0 2.8 3.0 6.8 2.9 3.4

0.48 0.16 0.07 0.36 0.11 0.22 0.04 0.15 0.12 0.62 0.83

a Six samples of cortical brain tissue from children of ages ranging from 1 year to 8 years.

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GM3 GM2

Fig. 7. Thin layer chromatogram showing the ganglioside pattern in a case of NCL of early onset (in the middle) and two normal controls. For details see text. Chloroform-methanol- 2.5 N N H 4 O H (60:35:8, all by vol.) was used as solvent.

ges even in the tetraglycosylganglioside group (GM1, GD 1a , GD l b, GT1), these normal main brain gangliosides were present in appreciable amounts. On many plates (Fig. 7) an additional spot containing neuraminic acid could be seen moving faster than GMx in solvents containing ammonia but slower in solvents containing water instead of ammonia. At present, the nature of this compound is unknown. Unfortunately it was not possible to estimate the total phospholipid content because of the limited amount of brain biopsy material available. DISCUSSION

A characteristic histological feature in our samples was the excessive occurrence in the nerve cell perikarya and glial cells of colourless to slightly yellowish autofluorescent granules which stained intensely with the PAS and Sudan black B methods and were acid fast and highly resistant to various lipid solvents. There was only slight or moderate distension of the neuronal perikarya and no true ballooning was seen. Both neurons and glial cells showed strong acid phosphatase activity in the cases assayed for this enzyme. These findings are consistent with the concept of so-called neuronal ceroid-lipofuscinosis as defined by Zeman and Dyken (1969). Biochemically our samples were characterized by a marked fall in the lipid-bound neuraminic acid, most pronounced in the older cases and probably reflecting the severe, progressive neuronal loss observed histologically. Thin-layer chromatography revealed a relative rise in the amount of N-acetylneuraminyl-lactosylceramide (GM3) in some cases, but normal

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main brain gangliosides were present in remarkable amounts. These findings, supported by morphological observations, exclude the known types of gangliosidosis. In addition to the general characteristics of NCL mentioned above our samples displayed some features considered typical of the J~insk2~-Bielschowsky type. Many neurons, not only in the basal ganglia and brain stem of the autopsy case but also in the cerebral cortex of most patients, contained irregular or spheroid inclusions corresponding to those described by Seitelberger (1962) and Seitelberger et al. (1967). Various transitional forms suggested that these inclusions were formed by coalescence from the usual granular storage material. The tinctorial properties of these inclusions differed clearly from those of Lafora bodies (Seitelberger 1968). Another feature shared with the J~insk2~-Bielschowskytype was the advanced cerebellar atrophy in our autopsy case. Not only the granule cells but also the Purkinje cells were totally and uniformly devastated in all parts of the cerebellum, a rim of PAS-positive phagocytes and hypertrophic astroglia indicating their previous site. The atrophy was not of the type usually associated with epilepsy (Peiffer 1963). In all of our patients ataxia appeared before the onset of epileptic seizures and frequently convulsions were not observed at all. This suggests that the cerebellar atrophy is probably due to the primary disease process and is not merely secondary to epilepsy. Many features in our cases do not fit in with any of the previously recognized forms of NCL. Even in our youngest patients there was a considerable cortical neuronal loss and in the older ones practically no nerve cells were visible in the cerebral cortex. This indicates a rapidly progressive neuronal destruction, despite the rather moderate storage process. Unusually severe cerebral neuronal loss has been observed in some similar cases (Brodmann 1914; Richter and Parmelee 1935; Bielschowsky 1936; Tingey, Norman, Urich and Beasley 1958; Hagberg, Sourander and Svennerholm 1968; Levine, Lemieux, Brunning, White, Sharp, Stadlan and Krivit 1968; Ryan, Anderson, Menkes and Denett 1970) and Zeman and Dyken (1969) suggest that it might be associated with the pronounced seizure activity. In many of our patients marked nerve cell destruction was established by cerebral biopsy before the onset of epileptic fits and therefore convulsions cannot be responsible for the neuronal loss in these patients. The involvement of the white matter seemed to be proportional to the cortical neuronal destruction, and was apparently related to the duration of the disease. The biopsies of older patients and the autopsy case were practically devoid of stainable myelin. Although some myelin degeneration is frequently encountered in NCL, lesions of this severity have so far been registered only in a few exceptional cases (Brodmann 1914; Richter and Parmelee 1935; Tingey et al. 1958; Hagberg et al. 1968 ; Levine et al. 1968; Ryan et al. 1970). A quite unusual characteristic of our cases was the tremendous cellular and fibrillary astrogliosis in the cerebral cortex present even in the youngest patient. The hypertrophic astrocytes had extremely coarse processes containing thick bundles of glial fibrils and thus resemble the giant astrocytes recently described by Hermann, Rubinstein and McKhann (1971). These findings contrast sharply with most previous observations of NCL where glial scar formation is said to be minimal even in the presence of severe neuronal loss (Zeman et al. 1970).

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The unique and dominating feature in our cases was, however, the presence o| great numbers of apparently phagocytic cells with coarsely granular cytoplasm and small hyperchromatic nuclei in the cerebral cortex of every patient. These cells were frequently bi- or even trinucleated and their number appeared to increase with the advancing neuronal destruction. This suggests that the cytoplasmic granules of these cells might be derived from phagocytosed material of decomposing neurons. The occurrence of phagocytic cells in cases of NCL has received little attention, although Seitelberger et al. (1967) and Zeman et al. (1970) mention the presence of occasional phagocytes in some of their cases. Massive occurrence of phagocytes, comparable to that found in our cases, has so far been reported only by Richter and Parmelee (1935), Tingey et al. (1958), Hagberg et al. (1968), Levine et al. (1968) and Ryan et al. (1970). The "ameboid" glial cells mentioned by Brodmann (1914) are possibly similar phagocytes. The fluorescence and the tinctorial properties of the granules contained in the phagocytes in our biopsies differed from those of the nerve cells. This might indicate that the material derived from decomposed neurons is subjected to some chemical transformation in the phagocytes. It is interesting to note that many of the phagocytes were multinucleated, a fact noted also by Tingey et al. (1958) who, however, interpreted these cells as modified nerve cells. This might give some clue as to the nature of the phagocytosed material, as multinucleated phagocytes have been produced experimentally in nervous tissue by certain types of glycolipids (Olsson, Sourander and Svennerholm 1966; Sourander, Hansson, Olsson and Svennerholm 1966). Various forms of cytoplasmic storage accumulations have been reported in electronmicroscopic studies of NCL. The most consistent type of storage body is the cytosome containing lamellar curved or semicircular membranes. These cytosomes with curvilinear profiles (Carpenter et al. 1972) (CC P) have been variously named in the literature (Zeman and Donahue 1963 ; Gonatas et al. 1968 ; Richardson and Bornhofen 1968 ; Duffy et al. 1968). According to a number of reports (Gonatas et al. 1968 ; Richardson and Bornhofen 1968 ; Duffy et al. 1968 ; Menkes and Price 1969; Elfenbein and Cantor 1969; Hermann et al. 1971; Ishii and Gonatas 1971; Carpenter et al. 1972) the CCP is the only type of storage body in many patients with the late infantile type of NCL, displaying their first clinical symptoms before the age of 4 years. In other cases usually classified as the juvenile type of NCL, the ultrastructural picture is more variable. As a transitional form of the previous type, storage bodies with curvilinear membranes mixed with "fingerprint" or some other type of material are occasionally found (Zeman et al. 1970; Hermann et al. 1971 ; Carpenter et al. 1972). In addition, various other structures of storage material have frequently been encountered (Gonatas, Terry, Winkler, Korey, Gomez and Stein 1963 ; Gonatas, Gambetti, Tucker, Evangelista and Baird 1969; Odor, Pearce and Janeway 1966; Sluga and Majdetzki 1967; Donahue, Zeman and Watanabe 1967; Hermann et al. 1971; Carpenter et al. 1972). The ultrastructure in these cases is thus more polymorphic and varies in detail in the individual patients. Some individual particles in these reports superficially resemble the uniform storage material in our cases (Gonatas et al. 1963 ; Sluga and Majdetzki 1967; Hermann et al. 1971) ; ultrastructurally typical lipofuscin bodies are also sometimes reported (Zeman et al. 1970; Gonatas et al. 1963).

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Our cases do not seem to belong to either of the groups mentioned above. The structure of the storage material is rather uniform consisting of small spherical globules of finely granular material and larger, usually membrane-bound conglomerations. The structure of the large bodies suggests that they are formed by fusion of the small globules (Fig. 4). No CCPs or other distinct structures were found. There are two recent reports on fine structure which possibly describe findings similar to ours. Ryan et al. (1970) studied formalin-fixed autopsy material, and the general pattern of their storage bodies is very much like ours. Levine et al. (1968) described the fine structure of storage material from a brain biopsy which resembled our biopsies, to judge from the electron micrographs. Thus, on clinical, histological and ultrastructural grounds, our 13 patients form a uniform group whose disease differs from the established types of NCL. As already noted in part I of this investigation (Santavuori et al. 1973), a few previous publications possibly refer to the same disease (Richter and Parmelee 1935; Tingey et al. 1958; Hagberg et al. 1968 ; Levine et al. 1968 ; Ryan et al. 1970). The disease is distinguished from other types of NCL by a clinical onset at around 1 year of age, a fulminant course with early retinal damage, absence or relative paucity of convulsions, severe neuronal destruction with massive occurrence of frequently multinucleated phagocytes and intense cortical fibrillary astrogliosis, and by the uniform granular ultrastructure of the storage material. Biochemical studies are now in progress to elucidate the basic metabolic disturbance in our patients and to establish their relationship to the very similar, if not identical, patient of Hagberg et al. (1968) with disturbed poly-unsaturated fatty acid metabolism.

ACKNOWLEDGEMENTS

We are greatly obliged to our colleague Dr. Kaarlo V. Parkkulainen who performed the brain biopsies, and to Dr. Risto Aine, Department of Pathology, Tampere Central Hospital, Finland, who made the autopsy and kindly placed the histological material at our disposal. We are indebted to Docent M~irta Donner, Helsinki, and Professor Patrick Sourander and Professor Bengt Hagberg, Gothenburg, Sweden, for valuable discussions.

SUMMARY

Morphological and biochemical data are given on brain biopsy or autopsy samples from 13 patients with a progressive encephalopathy, characterized clinically by the onset of rapid psychomotor deterioration at about 1 year of age, early amaurosis, and absence or relative paucity of convulsions. The main morphological feature was very severe neuronal destruction, accompanied by a massive occurrence of frequently binucleated phagocytes and unusually hypertrophic fibrillary astrocytes in the cerebral cortex. The remaining neurons and glial cells contained excessive amounts of autofluorescent granules with the staining properties of lipofuscin, and with strong acid phosphatase activity. The homogeneous granular ultrastructure of the storage material differed both from ordinary neuronal

284

M. HALTIA, J. RAPOLA, P. SANTAVUORI, A. KERANEN

l i p o f u s c i n a n d f r o m the s t o r a g e m a t e r i a l in m o s t p r e v i o u s r e p o r t s on s o - c a l l e d neuronal ceroid-lipofuscinosis. Biochemically the biopsy samples were characterized by a d e c r e a s e in l i p i d - b o u n d N - a c e t y l n e u r a m i n i c acid. T h u s , the u n u s u a l but u n i f o r m clinical, m o r p h o l o g i c a l a n d b i o c h e m i c a l f i n d i n g s in o u r series o f 13 p a t i e n t s differ f r o m t h e findings in p r e v i o u s l y r e c o g n i z e d t y p e s of a m a u r o t i c idiocy. T h e s e 13 p a t i e n t s - - a n d

p o s s i b l y s o m e o t h e r s c o l l e c t e d f r o m the

l i t e r a t u r e - - a p p e a r to c o n s t i t u t e a c l e a r l y s e p a r a b l e g r o u p " the r e l a t i o n s h i p of this c o n d i t i o n to v a r i o u s f o r m s o f n e u r o n a l c e r o i d - l i p o f u s c i n o s i s is discussed.

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