Glial Filaments Research Articles

Effects of age on the glial cells in the rhesus monkey optic nerve

The optic nerve is a circumscribed white matter tract consisting of myelinated nerve fibers and neuroglial cells. Previous work has shown that during normal aging in the rhesus monkey, many optic nerves lose some of their nerve fibers, and in all old optic nerves there are both myelin abnormalities and degenerating nerve fibers. The present study assesses how the neuroglial cell population of the optic nerve is affected by age. To address this question, optic nerves from young (4-10 years) and old (27-33 years) rhesus monkeys were examined by using both light and electron microscopy. It was found that with age the astrocytes, oligodendrocytes, and microglia all develop characteristic cytoplasmic inclusions. The astrocytes hypertrophy and fill space vacated by degenerated nerve fibers, and they often develop abundant glial filaments in their processes. Oligodendrocytes and microglial cells both become more numerous with age, and microglial cells often become engorged with phagocytosed debris. Some of the debris can be recognized as degenerating myelin, and in general, the greater the loss of nerve fibers, the more active the microglial cells become.

Infection of organotypic slice cultures from rat central nervous tissue with Neospora caninum: an alternative approach to study host–parasite interactions

Neospora caninum is an apicomplexan parasite which has emerged as an important cause of bovine abortion worldwide. Abortion is usually triggered by reactivation of dormant bradyzoites during pregnancy and subsequent congenital infection of the foetus, where the central nervous system appears to be most frequently affected. We here report on an organotypic tissue culture model for Neospora infection which can be used to study certain aspects of the cerebral phase of neosporosis within the context of a three-dimensionally organised neuronal network. Organotypic slice cultures of rat cortical tissue were infected with N. caninum tachyzoites, and the kinetics of parasite proliferation, as well as the proliferation-inhibitory effect of interferon-γ (IFN-γ), were monitored by either immunofluorescence, transmission electron microscopy, and a quantitative PCR-assay using the LightCycler instrument, respectively. In addition, the neuronal cytoskeletal elements, namely glial acidic protein filaments as well as actin microfilament bundles were shown to be largely colocalising with the pseudocyst periphery. This organotypic culture model for cerebral neosporosis provides a system, which is useful to study the proliferation, ultrastructural characteristics, development, and the interactions of N. caninum within the context of neuronal tissue, which at the same time can be modulated and influenced under controlled conditions, and will be useful in the future to gain more information on the cerebral phase of neosporosis.

Keratins as markers of epithelial cells.

AbstractEpithelial tissues, whether protective (such as skin or gut), or secretory (glandular linings) are required to withstand both physical and chemical stress, while at the same time maintaining normal tissue turnover. Much of this stress resistance is provided by a specialized cytoskeleton formed, as in all human cells, from three types of cytoplasmic filaments: actin filaments, microtubules, and intermediate filaments. The actin cytoskeleton is responsible for changes in cell shape and motility, the tubulin cytoskeleton for orientation and cytoplasmic polarization, and the intermediate filament cytoskeleton provides structural resilience (1). Intermediate filaments are 10-nm diameter cytoplasmic structures that, unlike the other two classes, show tissue-specific expression. The five main groups are the keratin filaments (epithelia), vimentin filaments (mesenchyma), desmin filaments (muscle), glial filaments (astrocytes), and neurofilaments (nerve).KeywordsAntibody DiluentKeratin FilamentBunsen BurnerFinal RinseKeratin ExpressionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Ciliary neurotrophic factor induces glial fibrillary acidic protein in retinal Müller cells through the JAK/STAT signal transduction pathway

Purpose. Intravitreal injection of ciliary neurotrophic factor (CNTF) is known to induce glial intermediate filament protein (GFAP) expression in retinal Müller cells. Because CNTF binding can activate multiple signaling kinases, we have examined the involvement of JAK/STAT pathway in GFAP induction in Müller cells. Methods. CNTF was injected intravitreally into mouse eyes. Immunocytochemistry and immunoblotting were used to study GFAP and STAT3-p (phosphorylated STAT3) levels either in mouse eyes, retinal explant cultures or in a Müller cell line, rMC-1. Results. In protein extracts of CNTF-injected eyes, retinal explants and the Müller cells, there was a substantial increase in STAT3-p level. Immunocytochemistry showed that STAT3-p was now present in many cell bodies in the INL and the GCL. To prove that CNTF acted via the JAK-STAT pathway, rMC-1 cells were transfected with a dominant-negative STAT3 mutant prior to treatment with CNTF. In the immunoblots of transfected cells, there was decrease in GFAP level. Conclusions. The results establish that CNTF can induce GFAP expression in retinal Müller cells through the JAK/STAT signaling pathway.

Development of astrocytes in the lamina cribrosa sclerae of the mouse optic nerve, with special reference to myelin formation.

In the mouse optic nerve, the optic nerve fiber layer in the retina, the optic papilla and the lamina cribrosa sclerae (LCS) just after penetrating the eyeball failed to generate myelin, whereas the optic nerve proper in the orbit was occupied by myelinated nerve fibers. The present study investigated development of the architecture of LCS, where the axons develop from unmyelinated to myelinated type, to elucidate how the initial part of axons was unmyelinated. At the LCS of the adult optic nerve, well developed astrocytes densely formed a cytoplasmic mesh-like frame through which unmyelinated fibers passed. The astrocytes here contained numerous and densely packed intermediate glial filaments and cell organelles. This framework formed by astrocytes appeared to be completed between 7 and 14 postnatal days before oligodendrocyte progenitors, migrated from the chiasm side, reached the proximal end of LCS, and began myelin formation. Thus the failure in myelin formation at the intraocular part and LCS possibly depended upon unsuccessful migration of oligodendrocytes beyond LCS constructed by specialized astrocytes, although other inhibitory factors for myelin formation, such as adhesion molecules distributed around LCS, may be unsolved.

Changes of Astrocytes in Retinal Ageing and Age-related Macular Degeneration

Most studies of age-related macular degeneration (AMD) have focused on the outer retina but little has been done on the involvement of astrocytes in this disease. We examined normal (young and old) and pathological (AMD) human retinas for the presence of changes in morphology and distribution of the astrocytes. Electron microscopy and inmunohistochemical techniques (anti-GFAP) were used for this study. Astrocytes in the ageing group showed: (1) higher GFAP immunoreactivity and more cytoplasmic organelles and glial filaments than astrocytes from younger retinas; (2) lipofucsin deposits; (3) a significantly smaller number of cells in the honeycomb astroglial plexus in the ganglion cell layer than in the younger group; and (4) Spaces with no GFAP reactivity in the nerve fiber layer. Changes observed in the AMD group were: (1) the basal membrane of the retinal capillaries was considerably thicker than in normal old individuals; (2) There were numerous non-functional capillaries; (3) There were hypertrophic astrocytes that phagocytosed dead ganglion cells; and (4) There were glial membranes constituted by astrocytes and Müller cells located between the vitreous humour and internal limiting membrane. These observations suggested that the extensive retinal ischaemia that can occur with AMD, together with the loss of astroglial cells accompanying normal ageing, could cause the death of the ganglion cells which cannot be protected from oxidative damage. Extensive ischaemia could cause the astrocytes to migrate to the vitreous humour where there is a metabolic reserve.

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice

Increased expression of γ-aminobutyric acid transporters GAT-1 and GAT-3 in the spinal trigeminal nucleus after facial carrageenan injections

The present study aimed to elucidate the distribution of γ-aminobutyric acid (GABA) transporters in the spinal trigeminal nucleus after carrageenan injections. Dense GAT-1 and GAT-3 but very little GAT-2 immunoreactivity was observed in the normal rat spinal trigeminal nucleus. The GAT-1-positive glial cells in the normal rat spinal trigeminal nucleus contained dense bundles of glial filaments and had features of astrocytes. Some GAT-3-positive cells contained dense bundles of glial filaments and had features of astrocytes, whilst others lacked glial filaments, and contained dense marginated heterochromatin, and had features of oligodendrocyte precursor cells. An increase in immunoreactivity to both transporters was observed on the injected but not the contralateral side 3 days after facial carrageenan injections. In rats given three further weekly injections of carrageenan and killed 3 days after the fourth injection, further increases in GAT-1 and GAT-3 immunoreactivities were observed. Electron microscopy showed that transporter immunoreactivity in the spinal trigeminal nucleus of carrageenan-injected rats was predominantly present in glial processes, showing that the increase in the number of processes observed at light microscopy was due to increased immunoreactivity in glial processes. An increased expression of GABA transporters in the carrageenan-injected spinal trigeminal nucleus could therefore result in a faster removal of GABA from the synaptic cleft of GABAergic axon terminals compared to normal rats. This could result in reduced inhibition/increased activity of the trigeminothalamic neurons in the spinal trigeminal nucleus, and could contribute to hyperalgesia after carrageenan injections.

Intermediate filament proteins define different glial subpopulations.

In certain species, specialized glial cells delineate cell domains in the central nervous system and assist in the elongation of axons by providing mechanical and chemical barriers. We showed previously, that the glial intermediate filament proteins vimentin and glial fibrillary acidic protein are extensively coexpressed in radial glia in the developing hindbrain, and that subsequently, the two proteins define distinct rhombomere domains: vimentin is localized in radial glia at the rhombomere boundaries and glial fibrillary acidic protein expression is restricted to the rhombomere centers (Yoshida and Colman [2000] J. Comp. Neurol. 424:47-57). The present study reveals that vimentin and glial fibrillary acidic protein continue to display distinct expression domains throughout the developing Xenopus central nervous system. Although the precise function of the two intermediate filaments in glial cells has yet to be revealed, the observations presented here suggests that glial intermediate filament proteins demarcate different populations of glial cells during nervous system development and that the existence of different glial populations may define glial boundaries.

Effects of Ionizing Radiation and Aluminum Chloride on Protein of Glial Intermediate Filaments in the Rat Brain

We studied the effects of irradiation with X-rays (the total dose of 0.0129 C/kg was attained over 7, 14, or 21 days), increased entry of Al3+ into the organism (0.2% AlCl3 in drinking water), and the combined influence of these factors for 21 days on the contents of the soluble and filamentous forms of glial fibrillary acidic protein (GFAP) in the tissues of the hippocampus, cerebellum, and neocortex of albino rats. After irradiation for 7 days, a clear trend toward drops in the GFAP contents in the structures under study was observed, while irradiation in the same dose, but for 14 or 21 days, resulted in increases in the contents of both GFAP forms (within a range of 13-29%, as compared with the control). Entry of aluminum chloride with water also resulted in an increase in the GFAP contents in all studied structures; changes in the filamentous form were more intensive. The combined influence of irradiation and Al3+ resulted in more intensive shifts in the GFAP levels; the content of its filamentous form increased in all structures by about 50%, while shifts of the soluble form were somewhat smaller.

Glial fibrillary acidic protein and vimentin immunoreactivity of astroglial cells in the central nervous system of adult Podarcis sicula (Squamata, Lacertidae).

The present immunoperoxidase cytochemical study describes the distribution of glial intermediate filament molecular markers, glial fibrillary acidic protein (GFAP) and vimentin, in the brain and spinal cord of the adult lizard, Podarcis sicula. GFAP immunoreactivity is abundant and the positive structures are mainly represented by fibres of different lengths which are arranged in a rather regular radial pattern throughout the CNS. They emerge from generally immunopositive radial ependymoglia and are directed from the ventricular wall towards the meningeal surface. The glial fibres give origin to endfeet which are apposed to the blood vessel walls and subpial surface where they form the continous perivascular and subpial glia envelopes, respectively. In the optic tectum and spinal cord, star-shaped astrocytes coexist with radial glia. In the spinal cord, cell bodies of immunopositive radial glia are displaced from the ependyma. While vimentin immunoreactive elements are almost completely absent in the brain except for a few diencephalic radial fibres, the spinal cord ependyma exhibits a clearly vimentin positivity and no GFAP staining. In the Podarcis CNS the immunocytochemical response of the astroglial intermediate filaments appears typical of mature astroglia cell lineage since it fundamentally expresses GFAP immunoreactivity. Moreover, this immunocytochemical study shows that the Podarcis fibre pattern with predominant radial glial cells is morphologically more immature than in avians and mammalians, a condition suggesting that reptiles represent a fundamental step in the phylogenetic evolution of vertebrate astroglial cells.

A light and electron microscopic study of the iron transporter protein DMT-1 in the monkey cerebral neocortex and hippocampus.

We have studied by immunocytochemistry, the distribution of DMT-1, a cellular iron transporter responsible for transport of metal irons from the plasma membrane to endosomes, in the normal monkey cerebral neocortex and hippocampus. Light to moderate DMT-1 staining was observed in glial cell bodies in the neocortex, the subcortical white matter, and the hippocampus. Despite light labeling of cell bodies, glial end feet around cortical and subcortical blood vessels were heavily labeled. In the neocortex, the glial cell bodies displayed the morphological features of protoplasmic astrocytes. Labeled glial cells in the subcortical white matter contained dense bundles of glial filaments and were identified as fibrous astrocytes. The observation that DMT-1 was present on astrocytic endfeet suggests that these cells are involved in uptake of iron from endothelial cells. It is possible that the iron could then be redistributed into the extracellular space in the brain parenchyma.

Development of Astroglial Elements in the Suprachiasmatic Nucleus of the Rat: With Special Reference to the Involvement of the Optic Nerve

The development of astroglial cells and the effect of the retinohypothalamic tract on it were studied by vimentin and glial fibrillary acidic protein (GFAP) immunocytochemistry in the suprachiasmatic nucleus (SCN) of the rat. At the embryonic stage, vimentin-immunoreactive (VIM-IR) radial glia, precursors of astrocytes, were dominant. However, their filaments vanished in the first few postnatal days. Instead of VIM-IR glial filaments, GFAP-immunoreactive (GFAP-IR) astrocytes appeared at E20 and grew rapidly from the P3 stage. GFAP immunoreactivity in the ventrolateral portion of the SCN (VLSCN) was measured using a computer-assisted image analyzing system. In normal rats, GFAP immunoreactivity showed a stepwise pattern with two slopes at P3–P4 and P20–P25. Bilaterally eye-enucleated rats operated on the day of birth showed lower GFAP immunoreactivity than normal rats and the GFAP immunoreactivity did not increase between P20 and P25 when GFAP-IR glial processes rapidly expand. Electron microscopic investigation at P50 (adult stage) revealed that neurons in the VLSCN had often direct apposition without astroglial processes and the frequency of this finding was significantly higher in eye-enucleated rats than in the control rats. These findings strongly suggest that the postnatal development of astroglial elements, particularly the expansion of GFAP-IR processes in the SCN, is regulated by retinohypothalamic projection.

Myocilin Expression in the Astrocytes of the Optic Nerve Head

We investigated the expression of myocilin in the optic nerve head of porcine eyes by Western blotting and immunohistochemical staining. Myocilin was localized in the nucleus, centrosome, glial filament, mitochondria, and some parts of the cell membranes of the astrocytes. Myocilin was also detected at the edge-feet portion of the processes of astrocytes adjacent to the inner limiting membrane and blood vessel wall. The astrocytes are the major cell population in the optic nerve head, contributing to the architecture of the nerve axon and blood vessels. Therefore, myocilin gene mutation and change of myocilin protein are likely to affect the architecture of the optic nerve head and induce various forms of glaucomatous optic nerve damage.

GFAP phosphorylation studied in digitonin-permeabilized astrocytes: standardization of conditions

Cycles of assembly/disassembly of the intermediate filaments of astrocytes are modulated by the phosphorylation of glial fibrillary acidic protein (GFAP). The sites on GFAP are localized at the N-terminal where they are phosphorylated by cAMP-dependent and Ca 2+-dependent protein kinases. Phosphorylation of GFAP has been investigated in brain slices, astrocyte cultures, cytoskeletal fractions and purified systems. Here we describe a different approach to study GFAP phosphorylation. We show that permeabilization of astrocytes in culture with digitonin allows direct access to the systems phosphorylating GFAP. Conditions for the permeabilization were established with an assay based on the exclusion of Trypan blue. Incubation of permeabilized cells with cAMP and Ca 2+ increased the phosphorylation state of GFAP. Immunocytochemistry with anti-GFAP showed that permeabilized astrocytes retained their typical flat, fibroblast morphology and exhibited well preserved glial filaments. On incubation with cAMP the filaments apparently condensed to form long processes. The results suggest the approach of studying structural changes in glial filaments in parallel to protein phosphorylation, in the presence of specific modulators of protein kinases and phosphatases has considerable potential.

A light and electron microscopic study of the GABA transporter GAT-3 in the monkey basal ganglia and brainstem.

The present study aimed to elucidate the distribution of the GABA transporter GAT-3 in the monkey basal ganglia and brainstem. Very dense GAT-3 immunoreactivity was observed in the medial septum, diagonal band, basal nucleus of Meynert, thalamus, globus pallidus, and substantia nigra. Moderate levels were observed in the subthalamic nucleus, periaqueductal grey, spinal trigeminal and vestibular nuclei. A general light level of staining was observed in the remainder of the brainstem regions, and very light staining was observed in the caudate nucleus and putamen. Electron microscopy showed that GAT-3 immunoreactivity was present in cell bodies with light cytoplasm and dense bundles of glial filaments, and features of astrocytes. Large numbers of astrocytic processes were also labeled in the neuropil. The cell bodies and processes of neurons were unlabeled. Further study is necessary to elucidate GAT-3 expression in neurological conditions, including hyperalgesia and Parkinson's disease.

Use of a heterologous monoclonal antibody for cloning and detection of glial fibrillary acidic protein in the bovine ventricular ependyma

From protozoans to vertebrates, ciliated cells are characterized by well-developed cytoskeletal structures. An outstanding example is the epiplasm, a thick, submembranous skeleton that serves to anchor basal bodies and other cell surface-related organelles in ciliated protozoans. An epiplasm-like cytoskeleton has not yet been observed in metazoan ciliated cells. In a previous study, we reported on MAb E501, a monoclonal antibody raised against epiplasmin-C, the major membrane skeletal protein in the ciliate Tetrahymena pyriformis. It was shown that MAb E501 cross-reacts with glial fibrillary acidic protein (GFAP), the class III intermediate filament protein found in astrocytes and other related glial elements. Here we used a post-embedding immunogold-staining method to localize MAb E501 cross-reactive antigens in ciliated cells from the ventricular ependyma in bovine embryos. When ependymocytes were treated with MAb E501, the ciliated region of the cell cortex was devoid of significant labeling. Instead, a gold particle deposit was evident around the nucleus, with only conventional ependymocytes being immunostained. Similar results were obtained by utilizing a rabbit antiserum against GFAP, revealing glial filaments and indicating an astroglial lineage of conventional bovine ependymocytes. In contrast, secretory ependymocytes of the subcommissural organ (SCO) were not stained by either of the two antibodies. Using MAb E501 as a heterologous probe, we cloned bovine GFAP cDNA. In situ hybridization experiments failed to detect GFAP transcripts in SCO ependymocytes, confirming the abscence of immunoreactivity in these cells.

Acidic calponin immunoreactivity in postnatal rat brain and cultures: subcellular localization in growth cones, under the plasma membrane and along actin and glial filaments.

Acidic calponin, an F-actin-binding protein, is particularly enriched in brain, where calponin protein and mRNA are mainly expressed by neurons. The presence of calponin immunoreactivity in cultured astroglial cells has been reported, but the presence of acidic calponin in astrocytes in vivo appears equivocal. For the present study, we raised a specific polyclonal antibody against the 16-residue synthetic peptide covering the sequence E311-Q326 (EYPDEYPREYQYGDDQ) situated at the carboxy-terminal end of rat acidic calponin, and we investigated the cellular and subcellular localization of the protein in the developing central nervous system. Our results show that acidic calponin is particularly enriched in: (i) growth cones and submembranous fields of maturing cerebellar and cortical cells, where it codistributes with microfilaments and (ii) glial cells in vivo, including radial glia, glia limitans, Bergmann glia and mature astrocytes, and ex vivo, where acidic calponin strongly colocalizes with intermediate glial fibrillary acidic protein (GFAP) and vimentin filaments. Finally, up to four acidic calponin subtypes with different isoelectric point (pI) values were identified by two-dimensional gel electrophoresis of cerebellar and hippocampal extracts. The more acidic isoforms were developmentally regulated. As only one single mRNA for acidic calponin has been identified, these isoforms must reflect postsynthesis changes probably related to the particular functions of acidic calponin in maturing cells. Although brain acidic calponin's exact role remains uncertain, the present data suggest that it is involved in neuronal and glial plasticity.

Taylor's cortical dysplasia: a confocal and ultrastructural immunohistochemical study.

In the present report we describe the neuropathological characteristics of tissue surgically resected from three patients affected by intractable epilepsy secondary to cortical dysplasia. Common features, suggestive of a focal cortical dysplasia of Taylor, were observed in all specimens. Immunocytochemical procedures were performed using neuronal and glial markers and the sections were observed at light traditional and confocal microscopes. This part of the investigation pointed out: 1. cortical laminar disruption; 2. very large neurons displaying a pyramidal or round shape; 3. ballooned cells; 4. decrease of calcium binding proteins immunoreactivity; 5. abnormal nets of parvalbumin- and glutamic acid decarboxylase-positive puncta around giant neurons but not around ballooned cells. Ultrastructural investigation on the same material provided evidence of a high concentration of neurofilaments in giant neurons and of glial intermediate filaments in ballooned cells. In addition, immunolabeled GABAergic terminals clustered around giant neurons were not found to establish synapses on their cell bodies. The present data, derived from a limited sample of patients but showing very consistent features, suggest that in Taylor's type of cortical dysplasia a disturbance of migratory events could be paralleled by a disruption of cell differentiation and maturation and by an impairment of synaptogenesis. This latter mechanism seemed to affect especially the inhibitory elements, and could account for the hyperexcitability of this tissue and thus for the high epileptogenicity of Taylor's dysplasia.

Induction of P-glycoprotein expression in astrocytes following intracerebroventricular kainate injections.

The expression of P-glycoprotein (PGP) was studied by immunocytochemistry and light and electron microscopy, in normal rats and after intracerebroventricular kainate injections. Two antibodies to PGP, mdr (Ab-1) and c-219, were used. As in previous studies (Thiebault et al. and Jette et al.), labelled capillaries were observed in normal rats. Kainate injections resulted in death of pyramidal neurons in the hippocampus, and a proliferation of glial cells in the affected cornu ammonis fields. An increase in PGP expression was observed in reactive astrocytes as early as 1 day postinjection. Immunoreactivity peaked at 2 weeks postinjection, but was still visible as late as 10 weeks postinjection. Similar results were observed using the two antibodies. Double immunolabelling and confocal microscopy also showed that PGP was colocalised with GFAP, a marker for astrocytes. The expression of PGP in astrocytes was confirmed by electron microscopy, which showed immunoreaction product in cells containing dense bundles of glial filaments and features of reactive astrocytes. The increased PGP expression in reactive astrocytes could be part of a cellular stress response program in these cells.