Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.     

Gap junctions between astrocytes during growth and differentiation in organ culture systems

Summary Fetal rat neocortex maintained in organ culture systems with the use of sponge foam matrices and millipore filter platforms undergoes growth and cytodifferentiation along classical neuronal and glial lines up to 36 days in vitro (DIV). Astrocytic differentiation is characterized by accumulation of 80–90 Å glial filaments in the cell bodies and processes of astrocytes. Gap or nexus junctions closely resembling those formed in mammalian brain in situ are identified by 15 DIV. By 36 DIV, interastrocytic gap junctions are numerous and frequently join extensive lengths of adjacent glial plasma membranes. The results suggest that these organ culture systems may provide a favorable environment for the study of cellular structure and function of coupled neuroglia.This work was supported by a research grant from the Veterans Administration. Skilled technical assistance was provided by Robin L. Isaacs and Marilyn Woodward     

Ultrastructural changes in neurons and neuroglia of the avian telencephalon (Hyperstriatum Accessorium) following tri-ortho-cresyl-phosphate intoxication

Summary Ultrastructural reactions of neurons of the avian forebrain following tri-ortho-cresyl-phosphate (TOCP) poisoning are described. These neurons show a marked increase in the rough endoplasmic reticulum (RER), with RER specializations such as lamellar bodies and subsurface cisternae, as well as a proliferation of the Golgi complex and neurofilaments. In addition, an increase in the number of dense bodies and mitochondrial osmiophilia is noted. Similar changes can also be observed in the neuroglia. These alterations appear 10–13 days after TOCP ingestion.     

Morphological studies on neuroglia

Summary The silver-impregnation procedure of Tsujiyama is suitable for demonstration of all three classical types of neuroglial cells; in the present study it was used for electron microscopic identification of neuroglial cells in the brain of the cat. The aim of the present study was 1) to determine impregnated structural correlates of neuroglial cells at the light- and electron-microscopic levels, and 2) to determine whether the method of Tsujiyama is applicable for the electron microscopic identification of the single types of neuroglial cells. Silver deposits were observed over the cytoplasm and processes of astrocytes where numerous glial filaments were present. Oligodendrocytes and microglial cells may be precisely differentiated by use of Tsujiyama's silver impregnation method at the electron microscopic level due to the pattern of silver-deposition in these two basic types of cells. This silver-impregnation method combined with electron microscopy is thus suitable for a precise identification of neuroglial cells; the technique may prove to be very helpful in identification of such categories of neuroglial cells that encompass also the images of cells which cannot be classified by use of the standard methods.Supported by a grant (No. 437002) from the Ministry of Education, Science and Culture, Japan     

Iron, Transferrin, and Ferritin in the Rat Brain During Development and Aging

Abstract: Iron is a universal cofactor for mitochondrial energy generation and supports the growth and differentiation of all cell types. In the CNS, iron is a key component of systems responsible for myelination and the synthesis of several neurotransmitters. In this study the spatial and temporal pattern of iron and its regulatory proteins transferrin and ferritin are quantitatively examined in the rat CNS during the first 3 weeks of postnatal life and in adults and aged animals. The midbrain, the cerebral cortex, and the cerebellum-pons are examined independently. Iron, transferrin, and ferritin concentrations are highest in all three brain regions at birth and decrease in each region to minimum levels during the third postnatal week. The decrease in levels of iron, transferrin, and ferritin is most pronounced in the cerebellum-pons and cortex and least in the midbrain. From postnatal day 17, iron (total iron content) and ferritin levels increase throughout the lifetime of the rat. In contrast, transferrin levels remain fairly constant in each brain region after postnatal day 24. The midbrain region, which includes the iron-rich regions such as the globus pallidus, substantia nigra, and red nucleus, has the least change in iron with development, has the highest level of ferritin during development, and consistently has the highest level of transferrin at all ages. These observations are consistent with reports that iron is important for normal motor function. Transferrin did not increase after postnatal day 24 in the three brain regions examined despite increasing amounts of iron, which implies a decrease in iron mobility in the aged rats, a finding that is consistent with observations of human brain tissue. The data reported in this study demonstrate that iron acquisition and mobilization systems in the CNS are established early in development and that the overall pattern of acquisition among brain regions is similar. These data offer support and insight into established concepts that a sufficient iron supply is critical for normal neurological development.     

大鼠中枢神经系统衰老的形态学研究:Ⅵ.大脑皮质胶质细胞的超微结构变化

对幼年、成年与老年Wistar大鼠大脑躯感皮质的胶质细胞的电镜研究表明:老年大鼠胶质细胞脂褐素增加和卫星化增多;在胶质与神经元胞体之间,神经元的胞质膜和胶质贴近处出现表面下复合器的情况也增多。星形胶质细胞还有胞质肥大和微丝增多的倾向。在个别老年动物中见到一种特殊的多层平行膜板状髓鞘样结构出现于胶质与神经元胞体之间。     

The astrocytes in the retina and optic nerve head of mammals: A special glia for the ganglion cell axons

Summary The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.     

Ultrastructure of the Lamellated Cytoplasmic Inclusions in Schwann Cells of the Marine Nematode,Deontostoma californicum

Electron microscopy of the photoreceptors in the marine nematode, Deontostoma californicum, revealed numerous lamellated inclusions in the Schwann cells ensheathing the lateral cephalic nerves. Immediately after the axons from the modified bipolar neurons of the photoreceptors enter the lateral nerves, these spherical-to-oval lamellated bodies are observed in the surrounding Schwann cell cytoplasm. These previously undescribed Schwann cell inclusions, approximately 500 nm long and 320 nm in diameter, are lamellated and characterized by the presence of an electron-dense stalk-like process, 80-280 nm long. The lamellated inclusions are bound by a single limiting membrane, 6-7 nm thick, which shows occasional interruptions. The internal structure of the inclusions is characterized by the presence of electron-dense lamellae or bands, 11-16 nm thick, which assume various complex patterns ranging from arrays of parallel linear densities to a reticulate appearance. In addition, the nematode Schwann cell cytoplasm contains the usual organelles, gliosome- and lysosome-like inclusions. Their relationship with lipofuscin pigments is briefly discussed.     

The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from 10-, 30- and 60-day-old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase-bright rounded cells having diameters after fixation of 7-12 μm. The contamination was primarily by red blood cells and phase-dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pg/cell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals.     

Initiating factors in perineuronal cell hyperplasia associated with chromatolytic neurons

Summary Neuronal hypertrophy and increased metabolism in nerve cells are evaluated as possible factors initiating hyperplasia of perineuronal cells. Colchicine induced neuropathy in the dorsal root ganglia is used as the model of increased neuronal metabolism.Twenty-eight female white rats weighing 100 g were divided into four groups, each animal receiving a 50 l injection into the subarachnoid space at the lumbosacral level eight days and again three days before sacrifice. The 50 l contained 25, 2.5 and 0.25 g of colchicine in distilled water for the first three groups and normal saline for the last group.A Zeiss ocular with random test points was used to determine the volume of tissue occupied by perineuronal cells and nerve cells in spinal ganglia. Direct cell counts yielded the size of the population of perineuronal cells and neurons.Irreversible motor and sensory loss occurred with the high dose injection, reversible loss with the 2.5 g injection and no loss with either the low dose or the saline injection. Chromatolytic neurons were noted in all animals receiving colchicine. Neither proliferation of perineuronal cells nor neuronal hypertrophy were observed. Neuronal hypertrophy, rather than altered neuronal metabolism, may be the initiating event in the perineuronal cell hyperplasia that frequently accompanies chromatolysis.