Barbara J. Wallace

Fine Structure of Spongy Degeneration of the Central Nervous System (van Bogaert and Bertrand Type)

The present study describes the fine structure of cortex and white matter of a one year old Jewish boy afflicted with spongy degeneration of the brain. The most characteristic clinical and pathologic feature of this disease is megalencephaly which is due to increased intracellular water content in the brain, mainly in the subcortical white matter. The light microscopic picture was similar to that previously reported. Electron microscopically, multiple vacuoles in the subcortical white matter and deep cortical lamina (fusiform layer) were due to separation of the lamellae of myelin. The vacuoles were present between the major dense lines of the myelin membranes, and it is conjectured that the obscured intraperiod lines might be the origin of these vacuoles. The extracellular spaces in the subcortical white matter were widened due to the rupture of the myelin lamellae and astrocytic cell membranes. It is hypothesized that a secondarily increased extracellular water content in the white matter might have accelerated further degeneration in the white matter at the advanced stage. There were smaller vacuoles present in the cortex, especially in the ganglionic layer which were the result of marked enlargement of astrocytes and their processes, although no increased extracellular spaces or ruptured cell membranes were present. The large and pale astrocytic nuclei described as Alzheimer type II by light microscopy appeared ultramicroscopically also enlarged and were seen to contain sparse nucleoplasmic granules, loss of chromatin and distinct nucleoli with preserved nucleolonema. Marked changes in size, and frequently in appearance, of mitochondria were observed in the processes of the astrocytes in the cortex, especially in the ganglionic layer. Histochemical studies showed that the activity of ATPase within these mitochondria was decreased when compared with that of normal brain tissue. The fine structure of the neurons, oligodendroglia and blood vessels was not altered.

Fine Structural Localization of Two Hydrolytic Enzymes in the Cerebellum of Children with Lipidoses

The fine structural localization of acid phosphatase (AcPase) and thiolacetate esterase activities were studied in biopsy tissue from the cerebellums of 2 children with Tay-Sachs disease (TSD), one child with late infantile amaurotie idiocy (LIAI), and one child with an acid mucopolysaccharidosis related to Hurlers disease (All). Localization of esterase was also studied in cerebellar tissue from a child with Niemann-Picks disease (NPD). In all cases AcPase and organophosphorus resistant esterase activities were localized largely in the abnormal lipid cytosomes characteristic of each disease. It is concluded that these lipid structures, which bear an apparent relationship to the endoplasinic reticulum, are lysosomes of the digestive or residual body type.

NUCLEOSIDE PHOSPHATASE AND THIAMINE PYROPHOSPHATASE ACTIVITY OF RABBIT GOLGI APPARATUS.

The Golgi associated nucleoside phosphatase which is demonstrable in rabbit pancreatic islet cells at alkaline pH using briefly postfixed cryostat sections and a modified Padykula-Herman incubation solution rapidly hydrolyzes uridine triphosphate (UTP) as well as uridine (UDP) and inosine diphosphate (IDP), but not thiamine pyrophosphate (TPP). Staining in the Golgi apparatus of islet cells was markedly attenuated when a lead incubation solution was employed at pH 7.2 with these same nucleoside-phosphates as substrates. The enzyme was not demonstrable in sections cut from tissue blocks fixed for 24 hours in formalin. This enzyme was distributed in the Golgi apparatus of some cellular elements of many other rabbit tissues. In rabbit cortical neurons a different Golgi enzyme was demonstrated which at alkaline pH hydrolyzed TPP but not UTP, IDP or UDP. This enzyme was also demonstrable with the lead method at pH 7.2 and under these conditions hydrolyzed IDP and UDP as well as TPP, but not UTP. The neuronal enzymatic activity was much less formalin-sensitive than that in pancreatic islet Golgi apparatus surviving in 24-hr formalin-fixed tissue blocks. This latter enzyme could be demonstrated in addition to brain only in the Golgi apparatus of hypophysis and epididymis. Differences in substrate specificity and of response to adjuvants as well as the differences in distribution confirm that these are two distinct enzymes.

Glial cell enzyme alterations in infantile amaurotic family idiocy (Tay-Sachs' disease).

~ N F A N T I L C amaurotic family idiocy (IAFI)? is characterized by neuronal swelling and degeneration due to deposition of gangliosides, by axonal degeneration and by marked reactive gliosis. In a recent histochemical study the authors repoitcd on the alterations of several oxidative enzymes and two phosphatases in ganglion cells of patients with IAFI (LAZARUS et aI., 1962h). It was found that oxidative en7yme activity was relegated to an area around the nucleus and just inside the cell nicmbrane, while acid phosphatase activity was greatly increased and was coextensive in the cell with the areas of accumulating lipid. From these findings and the evidence that the accumulated lipid forms laminated cytoplasmic bodies (TERRY et nl., 1962) it was hypothesized that the lipid is accumulated in lysosomes. The massive reactive gliosis seen particularly in the late phase of lAFt is due mainly to hypertrophy and proliferation of astrocytes and microglial cells, the latter of which also contain laminated bodies (TERRY et al., 1962) and show slightly different lipid staining qualities than the neurons (ARONSON et a/., 1955; DIPZEL, 1957; ARONSON and VOLK, 1962; TERRY ef nl., 1962). Numerous investigators have described increases in oxidative enzymes and acid phosphatases in reactive glial cells following trauma to the brain (RUBENSTHN et d., 1962; SMlltt and RUFENSTEIN, 1962; ANDERSON and SONG, 1962). These enzyme changes have been interpreted as reflecting the increased metabolism of the rapidly proliferating cells (RUBENSTEIN et al., 1962). Similar changes have been described in human neuropathological material including cases of oedema in the vicinity of brain tumours, inultiple sclerosis and cerebral arteriosclerosis with encephalomalacia (FRIEDE 1962). In view of our observations of enzymatic alterations in neurons of children afflicted with TAFI, it was of interest to determine to what extent the glial cell enzyme changes in this disease were a reflection of the proliferation and hypertrophy of glia and to what extent they were a peculiarity of the affliction itself. It was also of interest to determine whether or not the microglia which contain laminated bodies showed enzyme localizations similar to those seen in ganglion cells. In the present study, consideration is given to the localization of enzymes in central nervous system glial cells of patients with non-neurological diseases and of

HISTOCHEMICAL AND BIOCHEMICAL STUDIES OF GLOBOID CELL LEUCODYSTROPHY (KRABBE'S DISEASE)*

THE name ‘Krabbe type diffuse sclerosis’ has been applied to familial cases of diffuse sclerosis commencing in early infancy and characterized pathologically by the presence in white matter of large numbers of globoid cells which are non-metachromatic, non-sudanophilic and periodic acid Schiff (PAS) positive. Although histochemical staining methods suggest the presence of a n acid glycolipid in these cells (DIEZEL, 1957), biochemical studies have not as yet specifically identified the substance. BLACKWOOD and CUMINGS (1954) performed gross chemical analyses on the brain of a three-year-old child with Krabbe’s disease and found an increase of cerebrosides whereas other white matter lipids were decreased. On the other hand, more recent investigations (TINGEY, 1959; CUMINGS, 1960; AUSTIN, 1961, 1963; LEES and MOSER, 1962; and SVENNERHOLM, 1963) failed to reveal abnormal accumulations of cerebroside. AUSTIN (1961, 1963) reported an unequivocal decrease of total glycolipids, SVENNERHOLM (1963) a 50 per cent decrease of total lipid, and LEES and MOSER (1962) a decrease of total lipids including cerebroside but a relative increase of phosphatides. Although little is known concerning the specific biochemical alteration occurring in Krabbe’s disease, it is believed to be the result of an enzyme defect. Exceedingly limited information is available, however, concerning enzyme alterations in patients with this disease. AUSTIN (1961) reported high acid phosphatase activity in both human and experimentally induced globoid cells in rats. NELSON, AUREEECK, OSTERBERG, BERRY, JABBOUR and BORNHOFEN (1963) reported on a histochemical study of acid phosphatase and several dehydrogenases in Krabbe’s disease. To our knowledge no other reports on enzyme determinations in Krabbe’s disease exist. Recently the authors had the opportunity to study a case of Krabbe’s leucodystrophy occurring in a two-year-old girl. The histochemical localization of glucose 6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LD), and acid phosphatase (AcPase) were investigated, and biochemical determinations of LD and glutamic oxaloacetic transaminase (GOT) were made of tissue homogenates of both grey and white matter from various areas of the brain. In addition, determinations were carried out for total lipid and phospholipid. Lipid extracts of the brain of this patient as well as of brains of control patients were utilized for thin-layer chromatography.

LOCALIZATION OF PHOSPHATASES AND OXIDATIVE ENZYMES IN NEURAL TISSUE. EFFECT OF POST‐MORTEM AUTOLYSIS AND COMPARISON WITH CYTOLOGICAL LOCALIZATION IN TAY‐SACHS’DISEASE

anstieg von Enzymen des oxydativen Stoffwechsels feststellen. Im Friihstadium (innerhalb 24 Stunden) findet sich eine vermehrte Aktivitat der DPKund TPN-abhangigen Dehydrogenasen sowie der entsprechenden Diaphorasen ein. Zwei bis drei Wochen nach dem Trauma finden sich nur noch einzelne, aber immer noch sehr fermentaktive Axonkugeln ein. I n diesem Stadium sind die DPN-abhangigen Dehydrogenasen und die DPN-Diaphorase deutlich starker als die entsprechenden TPN-abhangigen Enzyme. Zur Klarung der Frage nach dem Zustandekommen des Enzymaktivitatsanstieges wurden am durchschnittenen peripheren Nerv Modellversuche unternommen. Dabei liess sich zeigen, dass der W E I S S ’ S C ~ ~ Axonstrom nicht die ausschliessliche Ursache fur derartige Veranderungen sein kann .