Overexpression of nestin and vimentin in ependymal cells in hydrocephalus

In order to elucidate the immunohistochemical features of hydrocephalic ependyma, immunohistochemical examination was undertaken in 11 normal, post-mortem brains (age range, 11 weeks’ postconception to 6 months after birth) and 12 hydrocephalic brains (three cases each of congenital aqueductal stenosis, Dandy-Walker malformation, Arnold-Chiari type II malformation and posthemorrhagic hydrocephalus) by using antisera to nestin, vimentin and glial fibrillary acidic protein (GFAP). In normal brains, nestin was predominantly expressed in neuroepithelial cells and radial glial fibers during the period of neuronal migration. Vimentin immunoreactivity was principally detected in immature ependymal cells and their basal fibers after the period of neuronal migration, then partly replaced by GFAP reactivity during late gestation. In hydrocephalus, the areas of ependymal disruption were covered with nestin- or vimentin-positive cells. Nestin and vimentin were also expressed in immature ependymal cells or their basal processes in anatomical regions such as the roof or floor plate of the fourth ventricle or the cerebral aqueduct, and the ventral part of the third ventricle. These results suggest that the overexpression of nestin and vimentin in hydrocephalus follows two patterns: a reactive pattern of proliferating immature glial cells associated with ependymal cell loss and an abnormal developmental pattern of immunopositivity associated with anatomical regions in the midline mesencephalon. Received: 27 November 1995 / Accepted: 29 December 1995     

Long-lasting coexpression of nestin and glial fibrillary acidic protein in primary cultures of astroglial cells with a major participation of nestin(+)/GFAP(-) cells in cell proliferation

Nestin, a currently used marker of neural stem cells, is transiently coexpressed with glial fibrillary acidic protein (GFAP) during development and is induced in reactive astrocytes following brain injury. Nestin expression has also been found in cultures of astroglial cells, but little is known about the fate and the mitotic activity of nestin-expressing cells in this in vitro model. The present study reveals a long-lasting expression of nestin in primary cultures of astroglial cells derived from the rat brain. Over 70% of the cells were nestin(+) at 12 weeks, with a large majority coexpressing the GFAP astrocytic marker. Time-course analyses supported a transition from a nestin(+)/GFAP(-) to a nestin(+)/GFAP(+) phenotype over time, which was further increased by cell cycle arrest. Interestingly, double staining with Ki67 revealed that over 90% of cycling cells were nestin(+) whereas only 28% were GFAP(+) in a population consisting of almost equivalent numbers of nestin(+) and GFAP(+) cells. These observations indicated that nestin(+)/GFAP(-) cells are actively engaged in mitotic activity, even after 2 weeks in vitro. Part of these cells might have retained properties of neural stem cells, insofar as 10% of cells in a primary culture of glial cells were able to generate neurospheres that gave rise to both neurons and astrocytes. Further studies will be necessary to characterize fully the proliferating cells in primary cultures of glial cells, but our present results reveal a major contribution of the nestin(+)/GFAP(-) cells to the increase in the number of astrocytes, even though nestin(+)/GFAP(+) cells proliferate also.     

Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord

Radial glial cell origins and functions have been studied extensively in the brain; however, questions remain relating to their origin and fate in the spinal cord. In the present study, radial glia are investigated in vivo using the neuroepithelial markers nestin and vimentin and the gliogenic markers GLAST, BLBP, 3CB2, and glial fibrillary acidic protein (GFAP). This has revealed heterogeneity among nestin/vimentin-positive precursor cells and suggests a lineage progression from neuroepithelial cell through to astrocyte in the developing spinal cord. A population of self-renewing radial cells, distinct from an earlier pseudo-stratified neuroepithelium, that resemble radial glial cells in morphology but do not express GLAST, BLBP, or 3CB2, is revealed. These radial cells arise directly from the spinal cord neuroepithelium and are probably the progenitors of neurons and the earliest appearing radial glial cells. GLAST/BLBP-positive radial glia first appear in the ventral cord at E14, and these cells gradually transform through one or more intermediate stages into differentiated astrocytes. Few if any neurons appear to be derived from radial glial cells, which are instead the major sources of astrocytes in the spinal cord. Evidence for the nonradial glial cell origins of some white matter astrocytes is also presented.     

Glial responses to neonatal hypoxic-ischemic injury in the rat cerebral cortex.

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic-ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.     

Sexual dimorphism in the hamster cerebellum demonstrated by glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity.

Male and female hamsters aged 1, 4, and 10 postnatal weeks were used to study the distribution of vimentin and glial fibrillary acidic protein (GFAP) in the cerebellum. Vimentin immunoreactivity exceeded that of GFAP during the first postnatal week, although GFAP was also observed in all cerebellar layers. Immunoperoxidase analysis revealed that by the fourth postnatal week vimentin was only detected in Bergmann fibers and the very scarce fibrous astrocytes located in the inner white matter. The Purkinje cell bodies were only coated with GFAP-immunopositive processes. At 10 weeks, vimentin immunoreactivity was reduced to thin Bergmann glial processes, whereas GFAP immunoreactivity had greatly increased in the whole cerebellum. The GFAP immunostaining was denser in males than in females; however, in females, the Bergmann fibers were heavily immunostained with anti-vimentin in contrast to the males. The results described in the present paper indicate a sex difference in vimentin and GFAP immunoreactivities in the cerebellar astrocytes at 4 weeks of age, which persisted in the oldest hamsters in this study. The existence of sexual dimorphism might suggest that the expression of both gliofilament proteins could be influenced by circulating sex steroids.     

Glial responses to neonatal hypoxic–ischemic injury in the rat cerebral cortex

Neurogenesis is nearly completed after birth, whereas gliogenic activities remain intense during the postnatal period in the developing rat cortex. These include involution of radial glia, proliferation of astrocytes and oligodendrocytes and myelin formation. Little is known about the effects of hypoxic–ischemic (HI) injury on these critical postnatal processes. Here we explored the glial reactions to mild HI injury of the neonatal rat cerebral cortex at P3. We show that the HI lesion results in disruption of the normal radial glia architecture, which was paralleled by an increase in GFAP immunopositive reactive astrocytes. The morphology of these latter cells and the fact that they were immunolabelled for both nestin and GFAP suggest an accelerated transformation of radial glia into astrocytes. In addition, BrdU/GFAP immunostaining revealed a significant increase of double-labelled cells indicating an acute proliferation of astrocytes after HI. This enhanced proliferative activity of astrocytes persisted for several weeks. We found an elevated number and increased mitotic activity of both NG2-positive oligodendrocyte progenitors and RIP-positive oligodendrocytes after injury. These findings imply that glial responses are central to cortical tissue remodelling following neonatal ischemia and represent a potential target for therapeutic approaches.     

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.     

Astroglial differentiation in the opossum superior colliculus

Glial markers, namely, vimentin, glial fibrillary acidic protein (GFAP), and glycogen, as well as accumulation of axon-borne horseradish peroxidase (HRP), were used to visualize radial glial cells in the developing opossum superior colliculus (SC) and to follow changes in young astrocytes of the superficial layers. Vimentin, GFAP, and glycogen are relatively abundant in elements of the median ventricular formation (MVF), which persists at least as late as weaning time, i.e., postconception day 103, postnatal day 90 (PND90). Radial profiles and end-feet in the remaining collicular sectors (main radial system, MRS) are also vimentin-positive but show little or no glycogen or anti-GFAP staining. The numeric density of MRS profiles is very high at the final stages of neuronal migration (PND12) but falls to vestigial numbers by PND 56-60. Antivimentin staining and filling of MRS profiles by axon-borne HRP disappear in parallel. Before total regression of MRS profiles, young astrocytes of the superficial gray layer exhibit a transiently high GFAP expression that is not found in those of the subjacent layers. The results suggest that 1) radial glia at or near the collicular midline are well equipped for a mechanical supportive role, and their abundant glycogen accumulation may reflect their eventual transformation in cells with high glycolytic metabolism, including tanycytes; 2) in most collicular sectors, some radial glia cells persist for long periods after cessation of neuronal migration and may interact with afferent fibers coursing through the superficial neuropil; 3) radially oriented astrocytes of the superficial gray layer exhibit a transiently high GFAP expression that is temporally correlated with late transformations of the retinocollicular projections.     

Intermediate filament protein synemin is transiently expressed in a subset of astrocytes during development

Synemin, a developmentally regulated protein first described in muscle cells, has recently been recognized as an intermediate filament (IF) protein. Because IF proteins are invaluable markers of cell origin within the nervous system, we were interested in determining the expression pattern of synemin in the brain. Our results show that, during development of the rat cortex, synemin is expressed only in a subpopulation of astrocytic cells expressing GFAP as well as vimentin and nestin. Unlike GFAP, however, synemin is not expressed in mature astrocytes and, unlike vimentin and nestin, synemin is not present in astrocytic precursors before GFAP expression. Taken together with morphological evidence, the time course of synemin expression, as determined by Western blotting, suggests that synemin is expressed in radial glial cells undergoing morphological transformation into astrocytes. Studies of synemin expression in vitro demonstrate that, early in primary culture, the majority of polygonal astrocytes are derived from synemin(+) radial glial cells. With time in culture, however, polygonal astrocytes either stop expressing synemin or are overgrown by cells not expressing synemin. The unique pattern of synemin expression, both in vivo and in vitro, suggests that the use of synemin as a marker will add a new dimension to studies of astrocytic differentiation.     

Prenatal exposure to ethanol alters the postnatal development and transformation of radial glia to astrocytes in the cortex

Postmitotic neurons migrate from a zone(s) near the ventricles to the neocortex. During this migration, neurons associate with radial glia. After serving their role as guides for neuronal migration, the radial glia transform into astrocytes. Prenatal exposure to ethanol causes abnormal neuronal migration. We examined the effects of gestational exposure to ethanol on radial glia and astrocytes. Radial glia were stained immunohistochemically with the antibody RAT-401, and astrocytes were labeled with an antibody directed against glial-fibrillary acidic protein (GFAP). The subjects were the offspring of rats fed an ethanol-containing liquid. diet (Et), pair-fed a liquid control diet (Ct), or fed chow and water (Ch). During the first postnatal week, radial glial fibers (in Et-treated rats and controls) stretched from the ventricular surface through the developing. cerebral wall to the pial surface. In the Et-treated rats, the radial processes were less dense and more poorly fasciculated than they were in the Ch-and Ct-treated rats. Moreover, by postnatal day (P) 5, there was a significant reduction in RAT-401 immunostaining in the Et-treated rats, particularly in the superficial cortex. A similar reduction in control rats did not begin until P10. In all three treatment groups, GFAP-immunoreactive astrocytes were in the cortex throughout the period from P1 to P45. In neonates, GFAP-positive cells were distributed in the marginal zone (layer I) and the intermediate zone (the white matter). The number of GFAP-positive cells in the cortical plate increased steadily with time so that, by P26, GFAP-immunoreactive astrocytes were distributed evenly through all cortical laminae. Interestingly, between P5 and P12, the number of astrocytes was significantly greater in Et-treated rats than in controls. Thus prenatal exposure to ethanol induces the premature loss of RAT-401-positive processes and the precocious increase in GFAP immunostaining. These ethanol-induced changes in glial development indicate that ethanol accelerates the transformation of radial glia into astrocytes. Moreover, the ethanol-induced premature degradation of the network of radial glial fibers may underlie the migration of late-generated neurons to ectopic sites. © 1993 Wiley-Liss, Inc.     

Effect of thyroid deficiency on the development of glia in the hippocampal formation of the rat: an immunocytochemical study

The development of glia in the hippocampal formation of normal and hypothyroid rats was studied using immunocytochemical staining for either glial fibrillary acidic protein (GFAP) or vimentin. Light microscopy showed lower GFAP immunoreactivity in the radial glial processes of young hypothyroid rats compared to normal animals. These processes followed the known path of neuroblast migration toward the proliferative zone of the dentate gyrus until the end of the 1st postnatal week. Vimentin immunoreactivity showed that the glial processes were present and therefore immature at least with respect to their cytoskeletal composition. We propose that this early defect in the maturation of the radial glial fibers accounts for the final deficit in the granule cells of the dentate gyrus. Later in development, thyroid deficiency also reduced the density and number of GFAP-labeled astrocytes and the growth of their processes. This observation is in complete disagreement with the glial hypertrophy induced by thyroid deficiency in the cerebellum. The considerably increased histogenetic cell death observed in the cerebellum of young hypothyroid rats could in turn induce glial hypertrophy, whereas the hippocampal formation, where a normal low number of cell deaths is observed, is only subjected to the general depressive effect of thyroid deficiency on cell maturation.     

Neurogenesis of heterotopic gray matter in the brain of the microcephalic mouse.

Neurogenesis of heterotopic gray matter in the brain of the microcephalic mouse prenatally exposed to X-rays at embryonic day 13 (E13) was studied immunohistochemically. Bromodeoxyuridine (BrdU) as a marker to label the migrating position of neuroblasts generated at various embryonic stages showed that no "inside-out" pattern of neuronal migration occurred in the heterotopic cell mass similar to that seen in the laminated cortex. Further results in which midkind (MK) immunoreactive radial glial fibers did not appear in the heterotopic cell mass demonstrated that heterotopia formed in the absence of radial glia system. Different types of cells (pyramidal and non-pyramidal neurons) in the heterotopic cell mass were identified with immunoreactivity for anti-parvalbumin and anti-calbindin D-28K antibodies in addition to current histological methods. Two major types of neurons were mixed together with random distribution in the heterotopic cell mass. This finding indicates that irradiation might have no selective effects on the precursors of pyramidal and non-pyramidal neurons. Moreover, anti-glial fibrillary acidic protein (GFAP) immunostaining showed that numerous astrocytes were present in the heterotopic cell mass. The fact that astrocytes appeared in the heterotopia without the transition from classic radial glial cells to astrocytes suggests that astrocytes might be generated directly from a separate astroglial precursor.     

Overexpression of nestin and vimentin in the ependyma of spinal cords from hydrocephalic infants

The ependyma of the spinal central canal in cases of hydrocephalus shows abnormalities which vary with the aetiology of ventricular dilatation. To determine whether these ependymal changes are developmental or reactive in nature, immunohistochemical findings were compared between nine normal controls and 12 cases of hydrocephalus (three each of congenital aqueductal stenosis, Dandy‐Walker malformation, Chiari type II malformation, and post‐haemorrhagic hydrocephalus) using antisera to nestin, vimentin and glial fibrillary acidic protein. The main pathological findings were disruption of ependymal layer, apparent pseudostratification of ependyma, expansion, cleft or syrinx formation in relation to the central canal, and ependymal rosette formation. In normal developing fetal spinal cord, nestin and vimentin were expressed mainly in pseudostratified ependymal cells and radial fibres in the median septum. In cases with congenital hydrocephalus (congenital aqueductal stenosis, Dandy‐Walker malformation, and Chiari type II malformation), nestin was overexpressed in immature ependymal cells, and strong vimentin immunoreactivity was detected in the long tract of radial fibres in the median septum. Nestin and vimentin were also expressed in small cells and their fibres which covered areas denuded of ependymal cells in cases of Chiari type II malformation and post‐haemorrhagic hydrocephalus. Two conclusions are suggested by this report. First, the ependyma of the spinal central canal in congenital hydrocephalus shows a delay in maturation of radial glial cells into mature astrocytes and ependymal cells. Second, areas of ependymal denudation may be repaired by the immature glial cells derived from subependymal cells.     

Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury

We have examined the injury response of astrocytes in the immature hamster brain in this study, focusing on alterations in the expression of glial fibrillary acidic protein (GFAP) and vimentin. In the adult CNS these two type III intermediate filament (IF) proteins have been shown to undergo robust increases in expression in response to axonal injury. Since injury to the immature CNS reportedly elicits less glial scar formation than adult brain injury, we examined the possibility that immature astrocytes respond differently than adult astrocytes to CNS injury with respect to IF gene expression. In situ hybridization using a ³⁵S-labeled cDNA GFAP probe was done on brainstem sections obtained 2,7 and 14 days after unilateral transection of the corticospinal tract in P8 hamster pups. The results indicated that substantial increases in GFAP mRNA were associated with the degenerating portion of the corticospinal tract by 2 days after axotomy and that the levels remained elevated for at least 14 days. Double-label immunofluorescence studies of this material suggested that GFAP as well as vimentin protein levels were also increased in many astrocytes in and around the degenerating corticospinal tract 2–14 days after axotomy. Most of the reactive astocytes in the degenerating regions exhibited increases in GFAP and vimentin immunostaining but some vimentin-negative GFAP-positive reactive astrocytes were also observed, particularly in regions surrounding the actual degenerative zones. The results from these experiments revealed that immature astrocytes have the potential for altering their normal developmental program of GFAP and vimentin expression after injury and mount a response that is qualitatively similar to that of astrocytes after CNS injury in the adult animal.     

GFAPδ immunostaining improves visualization of normal and pathologic astrocytic heterogeneity

Neuropathological analysis of cellular mechanisms underlying gliosis and brain tumors is slowed by the lack of markers allowing to distinguish glial subpopulations in normal or pathological human brains. We therefore evaluated GFAPδ immunostaining in a wide panel of astrogliosis and gliomas, and compared these with GFAP and vimentin. In normal tissue, gliosis and gliomas, GFAPδ immunostaining was observed in astrocytes with relatively high GFAP levels. GFAPδ immunostaining was most conspicuous in glia limitans astrocytes. In Chaslin's gliosis accompanying chronic epilepsy, GFAPδ immunostaining evidenced the glia limitans reactive astrocytes as the source of the dense fibrillary meshwork typical of Chaslin's gliosis. Interestingly GFAPδ and vimentin immunostainings coincided in normal tissues and gliosis, but not in gliomas. Altogether these results show that combined GFAP, GFAPδ and vimentin labelling reveals fine gliofilament regulation in normal and pathological brain.     

Developmental pattern of GFAP and vimentin gene expression in rat brain and in radial glial cultures

In the present study we analyze the events which occur during the early stages of astrogliogenesis by examining the pattern of both GFAP and vimentin gene expression and their corresponding immunoreactive proteins during rat brain development. This study was carried out “in vivo” (whole brain) and “in vitro” (primary culture of radial glia) using immunofluorescence, immunoblotting, and Northern blot analysis. Our results demonstrate that although GFAP immunostaining appeared late in gestation and at day 5 in radial glia cultures, GFAP mRNA expression was first detected, at very low levels, on fetal (F) day 15 and increased to F21. During postnatal development a striking increase in GFAP and its encoding messenger occurs. In contrast, the levels of vimentin and its mRNA expression were very high during the fetal stage (F15 to F21). Thereafter vimentin expression declined during postnatal (P) development until P21 and then remained constant at adult levels. In contrast, an increase in vimentin expression was observed in glial cells throughout the entire culture period. The biological significance of the developmental patterns of GFAP and vimentin expression in astroglial cells during brain development is discussed. © 1995 Wiley-Liss, Inc.     

Adult Nestin-expressing Subependymal Cells Differentiate to Astrocytes in Response to Brain Injury

The adult brain contains a small population of central nervous system (CNS) cells in the subependyma which, like embryonic CNS progenitor cells, express the intermediate filament nestin. In this report, the differentiation capacity in vivo of these cells was analysed following a standardized trauma. Before the trauma, the subependymal cells expressed nestin but not the astrocytic and neuronal differentiation markers glial fibrillary acidic protein (GFAP) and neurofilament respectively. In response to injury, the majority of the subependymal cells coexpressed nestin and GFAP, but never nestin and neurofilament. Furthermore, cells coexpressing nestin and GFAP were found progressively further away from the subependyma and closer to the lesion at later time points after the injury, indicating that these cells migrate towards the lesion. Nestin was in addition re-expressed in reactive astrocytes near the lesion and in non-reactive astrocytes very far from the lesion throughout the ipsilateral cortex. In conclusion, our data indicate that the nestin-positive subependymal cells are an in vivo source for the generation of new astrocytes but not neurons after injury, and that nestin re-expression in astrocytes following traumatic stimuli can be used as a sensitive marker for astroglial activation.     

Mab22C11 antibody to amyloid precursor protein recognizes a protein associated with specific astroglial cells of the rat central nervous system characterized by their capacity to support axonal outgrowth

Amyloid precursor protein (APP) is a transmembrane glycoprotein which is believed to promote neural cell adhesion, neural survival, and neuritogenesis. The present study was undertaken to determine whether APP could be detected within different types of astroglial cells present in the central nervous system (CNS) of neonatal or adult rats. The localization of this protein within glial cells was studied by using a monoclonal antibody (Mab22C11) that recognizes all APP isoforms and in addition cross-reacts with APP-like proteins. In the brain of neonatal rats, Mab22C11 immunostaining was associated with numerous elongated radial glia-like structures. In the intact brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with (i) numerous neuron-like structures and (ii) glial structures immunostained for glial fibrillary acidic protein (GFAP) and/or vimentin, including tanycytes mostly located in the mediobasal hypothalamus, fibrous astrocytes located in the white matter and ependymocytes bordering the ventricles. On the other hand, all the GFAP-immunostained astrocytes located in the grey matter were Mab22C11 negative. In the lesioned brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with intensely GFAP-immunostained reactive astrocytes located close to a surgical lesion, but not with those induced by Wallerian degeneration that appear at a distance from a lesion. Electron microscopic observations further indicated that in all these labeled astroglial cells, Mab22C11 immunostaining was mainly localized to the limiting plasma membrane and the membrane of intracytoplasmic cisternae and vesicles. These data indicate that Mab22C11 antibody induces strong immunostaining of specific astroglial cells of the neonatal and adult rat CNS that support axonal outgrowth, therefore suggesting that an APP-like protein associated with these cells participates in their axonal outgrowth promoting properties. J Comp Neurol 377:550–564, 1997. © 1997 Wiley-Liss, Inc.