The earliest alterations in rat neurones and astrocytes after anoxia-ischaemia

Summary A combined light and electron microscope study was made of the alterations occurring in the neurones and astrocytes of the neocortex and hippocampus of rats killed immediately after intermittent exposures to nitrogen of 5 and 15 min. Blood flow in the right common carotid artery had previously been interrupted by application of an artery clasp which was removed after the exposure to nitrogen and the animals killed by perfusion-fixation with glutaraldehyde.Microvacuolation (MV), the earliest stage of anoxic-ischaemic neuronal damage, was observed in the ipsilateral neocortex and hippocampus of both groups and ischaemic cell change (ICC) bilaterally in the neocortex of animals exposed for 15 min. Ultrastructural examination showed the microvacuoles to be swollen mitochondria.Slightly dense, mildly distorted, non-vacuolated neurones were also seen in the neocortex and hippocampus. They did not exhibit the ultrastructural changes seen in MV and ICC.Swollen astrocytic processes were sometimes seen around the damaged neurones, more frequently after 15 min exposure. Slight swelling of perivascular astrocytic processes was occasionally observed while the extracullular spaces in the neuropil remained unaltered. This implies that the accumulation of fluid in oedematous grey matter is confined to the astrocytic compartment.The reversibility or otherwise of all the neuronal alterations is discussed.     

Status spongiosus of nervous tissue

Summary The diverse nature of status spongiosus of nervous tissue is revealed by electron microscopy in a variety of experimental models. Intracellular, extracellular and intramyelinic accumulation of fluid as well as artefactitious dissolution of myelin may account for spongiform changes. In cerebral edema following trauma the perivascular and perineuronal vacuoles in the cortex correspond to swollen astrocytic processes whereas in the white matter the sponginess mainly reflects enlarged extracellular spaces. Vacuolation of swollen dendrites, termnal axons and perikaryal cytoplasm of nerve cells characterizes the transmissible spongiform encephalopathies. Axonal swelling produced by anoxia accounts for the spongy changes seen in the optic nerve in acute glaucoma. Selective accumulation of fluid within myelin sheaths occurs in isonicotinic acid hydrazide poisoning. Aggregates of irregularly curved, lamellar structures, derived from the artifactitious disintegration of myelin, form grape-like deposits or Buscaino bodies which, when dissolved, appear as vacuoles that may be confused with other spongiform states.This study was supported in part by United States Public Health Research Grant NS-09053 from the National Institute of Neurological Diseases and Stroke, NIH, Bethesda, Maryland.     

SPONGIFORM POLIOENCEPHALOMYELOPATHY CAUSED BY A MURINE RETROVIRUS. II. ULTRASTRUCTURAL LOCALIZATION OF VIRUS REPLICATION AND SPONGIFORM CHANGES IN THE CENTRAL NERVOUS SYSTEM

The development of murine retrovirus induced spongiform polioencephalomyelopathy was studied sequentially by electron microscopy. During the initial 30 days, viral infection of the central nervous system, as evidenced by viral budding from membranes, was limited to the endothelial cells and pericytes. Viral particles were observed in the lumen of blood vessels, extracellular spaces and astrocytic endfeet surrounding blood vessels, but no morphological evidence of productive infection was found in astrocytes or neurons during early development of vacuolation. The earliest lesions in the neuropil consisted of swelling of astroglia followed by vacuolation, initially in axons and dendrites and later in neuronal and astrocytic soma, where vacuoles appeared to arise from dilated cisternae of the Golgi apparatus. Vacuoles contained only amorphous debris and fragments of membranes. Virions budding aberrantly into vacuoles were seen only in mice surviving beyond 35 days. Numerous reactive astrocytes were observed, but inflammatory cells were absent. The ultrastructural changes were remarkably similar to those described in scrapie, Kuru, and Creutzfeldt-Jakob disease.     

Serial ultrastructural study of experimental Creutzfeldt-Jacob disease in guinea pigs

Summary Guinea pigs inoculated with brain homogenate from serially passaged Creutzfeldt-Jakob disease (CJD) were killed biweekly starting at week 2 until terminal illness (about 200 days following inoculation). A mild swelling of postsynaptic dendrites and an increase in the number of glial filaments in astrocytic processes was seen at week 4, followed by increased swelling and lucency of axons and dendrites by week 6 post inoculation (p.i.). Severe undulation and focal interruptions of synaptic membranes were also observed both at weeks 4 and 6. By week 8, one could see cystically dilated cellular processes. These sometimes showed continuity with adjacent swollen processes through focally disrupted plasma membranes, and most likely represent a progressive enlargement of vacuoles through fusion and subsequent addition of adjoining processes. The spongiform changes increased mildly between week 8 and week 10 and remained essentially the same in subsequent weeks. After week 24 there was a sharp increase in both the number and size of vacuoles. At week 24 severe structural alterations were present both in the neurons and astrocytes, and numerous intranuclear inclusions were demonstrated in many neuronal nuclei. This study shows that morphological changes in the brain occur considerably earlier than the clinical manifestations of the disease. In the early phase of the disease, there were significant alterations on the dendrites and synapses.Supported by grants NS 12674 and AG 03106 from the National Institutes of Health     

Electron miscroscopic and enzyme histochemical studies of the cerebellum in spongy degeneration

Summary Electron microscopic and enzyme histochemical studies were performed on the cerebellum from a 9 month old Jewish boy with spongy degeneration. Histologically, the main pathological changes were noted in the Purkinje cell layer, the deeper areas of the granular cell layers and the subcortical white matter. Ultrastructurally, multiple vacuoles were present within the swollen cytoplasm and processes of protoplasmic astrocytes in the cortex, while in the subcortical white matter vacuoles were observed within splitting myelin lamellae as well as within astrocytes. There were also abnormal mitochondria within swollen protoplasmic astrocytic cytoplasm and processes which in ATPase preparations showed little or no reaction product. However, the fibrillary astrocytes were not swollen and contained intact mitochondria which showed normal reaction product in ATPase preparations. Since the myelin changes are known to be nonspecific and secondary to abnormal fluid accumulation, the characteristic distribution of the multiple vacuoles in the central nervous system in this disorder seems primarily to be related to swelling of the protoplasmic astrocytes.Supported by a grant from the National Tay-Sachs and Allied Diseases Association, Inc.     

ENHANCED PERMEABILITY TO HORSERADISH PEROXIDASE ACROSS CEREBRAL VESSELS IN THE RAT AFTER PORTOCAVAL ANASTOMOSIS

The blood—brain barrier to horseradish peroxidase (HRP) was examined in rats 10 or 30 days after establishing an end-to-side portocaval anastomosis. An increased number of vesicles containing HRP were seen in the endothelial cells of arterioles and to a lesser degree in the endothelial cells of venules and capillaries. Peroxidase was not found in the interendothelial space between tight junctions, which were everywhere intact. The basement membranes of vessels and the neuropil adjacent to these could contain the tracer. Astrocytic endfeet were swollen 10 days after the operation and contained only a few organelles. Thirty days after the operation reactive changes seemed to occur in astrocytes, indicated by less swelling and enhanced number of organelles. The cytoplasm of the astrocytes in some instances was evenly stained by reaction product. A similar staining was very occasionally observed in a few neuronal processes. It is concluded that an increased vesicular transport of HRP is inferred to have occurred across cerebral endothelium in animals after PCA and that the astrocytes may be especially sensitive to damage to the plasma membrane during liver shunt encephalopathy leading to intra-cellular uptake of substances from the extracellular space.     

Scrapie inoculation of mice: light and electron microscopy of the superior colliculi

Summary Ultrastructural examination of the superior colliculi of mice intraocularly inoculated with the ME7 strain of scrapie showed vacuolation early in the course of infection. Brains were examined between 85–260 days after monocular inoculation with scrapie. The mean incubation period for the development of clinical disease was 302 days. Vacuolation was seen initially in the contralateral superior colliculus and subsequently in the ipsilateral colliculus. In coded trails light microscopical vacuolation was seen from 218 days but ultrastructural examination showed that sparse vacuoles were inconsistently present in either or both of the ipsilateral and contralateral colliculi from 85 days; frequent vacuoles were seen from 190 days. Scrapie-induced vacuoles were differentiated from vacuoles present in control tissue by the presence of loculation or by a limiting double membrane which showed protrusion or proliferation of the innermost lamella. Vacuolation was seen in neuronal perikarya, myelinated fibres, dendrites and axonal presynaptic terminals. Vacuoles of myelinated fibres were observed within myelin and possibly also in the inner tongue of oligodendroglial cytoplasm. Whorled membrane configurations were also seen. Tubulovesicular particles, 40 nm in diameter, were recognised in two scrapie-infected mice. It is suggested that some scrapie vacuoles arise as a result of incorporation of abnormal membrane into organelles, possibly mitochondria, in neuronal perikarya and neurites and probably also within oligodendroglial cytoplasm and myelin.     

The Golgi apparatus-complex of neurons and astrocytes studied with an anti-organelle antibody

An antiserum reacting with a 135-kDa antigen of rat liver Golgi apparatus-complex was used to stain, by light microscopic and ultrastructural immunocytochemistry, sections of rat cerebellum and by immunoblot homogenates of whole brain, isolated neurons and a fraction of enriched neuronal Golgi apparatus. In sections of rat cerebellum fixed with periodate-lysine-paraformaldehyde and immunostained with the direct peroxidase or peroxidase-antiperoxidase methods, the Golgi apparatus-complex in perikarya of neurons and glia was stained. Occasionally, nuclear envelopes and cisternae of the rough endoplasmic reticulum of neurons and glia were stained. Immunostain was not observed in peripheral dendrites, axons and presynaptic terminals. In striking contrast, peripheral smooth cisternae of astrocytic perikarya and processes were stained. Immunoblots of whole-brain membrane fractions, homogenates of isolated neurons and an enriched neuronal fraction of the Golgi apparatus-complex showed a principal single band of 64-kDa apparent mol. wt. We have concluded that the putative 64-kDa antigen(s) is distributed in cisternae of the Golgi apparatus-complex and occasionally in the nuclear envelope and rough reticulum, within the perikarya of neurons and glia. A second important distribution of the 64-kDa antigen(s), involving peripheral cisternae in perikarya and processes of astrocytes, is consistent with the hypothesis that the Golgi apparatus-complex of these cells extends to the periphery of these cells. The functional implications of the peripheral localization of the 64-kDa antigen(s) in astrocytes are discussed.     

NEURONAL CHANGES IN EXPERIMENTAL GLIOMAS

Gliomas were induced transplancentally by the administration of ethylnitrosourea to pregnant rats on the 15th day of gestation. The fine structure of neurones involved in these tumours was studied in order to assess the changes caused by neoplasia. The severity of the neuronal damage depended on their location, Those within the tumours being more affected than those in the neighbouring brain. The histological type and grade of malignancy also influenced the changes: periventricular pleomorphic gliomas and ependymomas of high-grade malignancy caused the most severe alterations. Neurones displayed a spectrum of appearances from apparent normality to complete necrosis. Both nucleus and cytoplasm were affected: of the cytoplasmic organelles the rough-surfaced endoplasmic reticulum and mitochondria showed the most striking changes. There were many lipofuscin granules and autophagic vacuoles. Axons underwent advanced degeneration: swollen mitochondria, disrupted vesicles and vacuoles, disintegrated microtubules, irregular filaments and unidentified debris were sometimes seen. Disintegration of myelin sheaths frequently occurred with consequent engulfing of their debris by macrophages and reacting astrocytes. The neuronal satellites--mainly oligodendrocytes--were often replaced by neoplastic glial cells. Hydrolytic 'marker' enzymes were demonstrated in neurones at various stages of degeneration. In all cases neurones displayed low acid phosphatase activity, while thiamine pyrophosphatase activity decreased with increasing neuronal degeneration.     

Neuronal autophagic vacuoles in experimental scrapie and Creutzfeldt-Jakob disease

Summary We report the presence of autophagic vacuoles (AV) in neuronal perikarya and neuronal processes of rodents with experimental scrapie and Creutzfeldt-Jakob disease. AV were composed of sequestrated cytoplasmic areas containing ribosomes and occasionally mitochondria and small secondary vacuoles. The formation of AV may contribute to neuronal degeneration and ultimately to neuronal loss.P. P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Polish Academy of Sciences (VIII/40)     

Ultrastructural findings in experimental Creutzfeldt-Jakob disease in guinea pigs

Brains from six clinically ill guinea pigs from various serial passages infected with brain homogenate from a patient with Creutzfeldt-Jakob disease were studied by light and electron microscopy. There were no appreciable morphologic differences among the animals at different passages. Light microscopy revealed clearing, swelling, and vacuolar changes in neurons and astrocytes. These changes occurred in both perikarya and cell processes. Large isolated vacuoles in the neuropil appeared to develop by progressive fusion between swollen cellular processes. By electron microscopy many neuronal processes contained degenerating organelles, osmiophilic bodies, membranous bodies and approximately 10 mm filamentous structures. In addition, vacuoles and membranous inclusions were seen in some neuronal nuclei, changes which have not been previously observed in spongiform encephalopathies.     

Insect brain as a model for the study of aging

Summary Behavioral, light and electron microscopic studies of young and oldDrosophila melanogaster are presented. Negative geotaxis and mating studies were used as indices of behavior; these began to decline at ages 28–35 days and very low values were obtained at 70 to 85 days. With the light microscope the brains of the old flies demonstrated shrinkage of the cortex, loss of basophilia in the cytoplasm of the giant neurons (neurosecretory cells), and vacuolation in the neuropil. Electron microscopy of the brains of aged flies revealed a marked loss of free ribosomes and granular endoplasmic reticulum and increased cytoplasmic lipid and autophagic vacuoles in neurosecretory cells. There was also a general decrease in the amount of neuronal cytoplasm. Vacuoles and pale processes, sometimes identified as swollen cell processes, were frequently seen in the deep cortex and neuropil. A heterogeneous population of dense bodies was found in the neuropil. Morphologically the bodies resemble lipoprotein complexes and are consistent with age pigment, but further characterization awaits biochemical studies.Supported in part by Research Grant NS-08276 from the National Institute of Neurological Diseases and Stroke, U.S. Public Health Service. Presented in part at the 46th Annual Meeting of the American Association of Neuropathologists, June 12–14, 1970, Atlantic City, N.J.     

Marek's disease virus-induced transient paralysis in chickens: electron microscopic lesions

Summary A study was made to determine the causative lesion of Marek's disease virus (MDV)-induced transient paralysis (TP) in chickens by comparing the ultrastructure of brain tissue from MDV-infected genetically susceptible and resistant birds. There were numerous intramyelinic vacuoles in the brains of TP-affected birds. Many of these vacuoles contained particulate material compatible with precipitated protein from edema. Astrocyte processes often were distended with similar particulate material. Most intrameylinic vacuoles were either adjacent to the axolemma or within inner myelin lamellae. Myelin sheaths of affected axons, while being displaced, were relatively normal with no vesiculation. Most affected axons were also otherwise normal. Cell processes adjacent to occasional affected axons were distended by degenerating mitochondria, vacuoles, and amorphous material. Some of these processes appeared to be inner loops of oligodendrocyte cytoplasm. The cell bodies of most oligodendrocytes were normal, but a few contained vacuoles similar to those seen in processes adjacent to axons. There were scattered necrotic cells. While most of these could not be specifically identified, some appeared to be oligodendrocytes. Mononuclear inflammatory cells were present both perivascularly and within the parenchyma. Although these cells occasionally contacted myelinated axons and there was myelin phagocytosis, there was no indication that they initiated demyelination. Brain tissue from virus-inoculated resistant birds had perivascular aggregates of mononuclear cells, but there were no intramyelinic vacuoles and few necrotic cells. These findings suggest that intramyelinic vacuolation contributes to the pathogenesis of transient paralysis. Potential pathophysiological mechanisms contributing to the vacuoles, including brain edema and oligodendrocyte injury, are discussed.Supported in part by the National Institute of Neurological and Communicative Disorders and Stroke, NIH, Grant No. NS16770 and by the State of North Carolina     

Subacute spongiform encephalopathy: Electron microscopic studies

Electron microscopic changes in brain biopsies from two cases of subacute spongiform encephalopathy are described. Large vacuoles seen in astrocytic and neuronal processes were surrounded by one or more concentric membranes. Dense-core vesicles were found in axonal endings and in nerve cell perikarya; these particles could represent catecholamine-containing vesicles.     

A light- and electron-microscopic study of a temporal-lobe ganglioglioma

A temporal-lobe ganglioglioma was surgically removed from a 13-year-old boy who had psychomotor seizures for 4 years. By light microscopy, the ganglion cells were encompassed in a rich network of fibrous connective tissue. They merged with areas composed of sparser neuronal cells and numerous fibrillary astrocytes. The ganglionic and astrocytic areas contained many tiny eosinophilic granules, and proteinaceous globules which measured from 1 to 10 μ and often formed circular aggregates measuring up to 60 μ. By electron microscopy, three main cell types were seen:

1.

(1) Ganglion cells, whose cytoplasm and processes frequently contained dense-core vesicles measuring from 1250 to 1800 Å. These cells were sometimes found in synaptic contact with adjacent processes. The dilated nerve cell processes often contained concentrically laminated bodies resembling those reported in experimental axonal reaction and degeneration. Some of these bodies may represent degenerative changes in the dense-core vesicles. Larger autophagic vacuoles, occasionally containing a dense-core vesicle, were also found.     

Cell death and autophagy in prion diseases (transmissible spongiform encephalopathies)

Neuronal autophagy, like apoptosis, is one of the mechanisms of programmed cell death. In this review, we summarize current information about autophagy in naturally occurring and experimentally induced scrapie, Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker syndrome against the broad background of neural degenerations in transmissible spongiform encephalopathies (TSEs). Typically a sequence of events is observed: from a part of the neuronal cytoplasm sequestrated by concentric arrays of double membranes (phagophores); through the enclosure of the cytoplasm and membrane proliferation; to a final transformation of the large area of the cytoplasm into a collection of autophagic vacuoles of different sizes. These autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. On the basis of ultrastructural studies, we suggest that autophagy may play a major role in transmissible spongiform encephalopathies and may even participate in the formation of spongiform change.     

Alterations of astrocytic organelles in various lipidoses and allied diseases

Summary The astrocytic organelles in 12 patients with various lipidoses and allied diseases were studied. Histochemically and ultrastructurally, astrocytes contained cytoplasmic inclusions which were identical to those seen in neurons from cases of similar disorders. In the early stage of these disorders, the astrocytic cytoplasm displays a close relationship between increased numbers of mitochondria and dilated smooth portion of the endoplasmic reticulum. In the more advanced stage, the mitochondria decrease in number, and the lipid bodies show close proximity to or continuity with the smooth portion of the endoplasmic reticulum. During the late stages of the disorder, the intracytoplasmic inclusion bodies become numerous, and the mitochondria are further reduced in number. It, therefore, appears that in astrocytes in these disorders the mitochondria and the smooth portion of the endoplasmic reticulum play an important role during the evolution of the intracytoplasmic lipid bodies. On the basis of these observations, the possible morphogenesis of lipid cytosomes is discussed.Supported by grants from the National Institutes of Health (B-2977) and the National Tay-Sachs and Allied Diseases Association, Inc.     

αB-Crystallin negative astrocytic inclusions

We report on an unusual pathological finding of astrocytes, observed in the brain of a 16-year-old African-American male with severe intellectual disability and spastic quadriplegia. The brain showed bilateral pericentral, perisylvian polymicrogyria and pachygyria, in conjunction with a large number of hypertrophic astrocytes with eosinophilic granular cytoplasmic inclusions. The astrocytic abnormality was more severe in the dysgenetic area but present throughout the cerebral cortex. Astrocytic inclusions stained with acid fuchsin, azocarmine and Holzer’s stain, and were immunoreactive for GFAP, S-100, and ubiquitin, but not for αB-crystallin, filamin, vimentin, nestin, tau or α-synuclein. Based on the case and a review of the literature, the authors postulate that these astrocytic inclusions in the cerebral cortex reflect abnormalities in radial glial developmental processes, such as migration, differentiation, or glial-neuronal interaction function during neuronal migration.