Neuritic outgrowth associated with astroglial phenotypic changes induced by antisense glial fibrillary acidic protein (GFAP) mRNA in injured neuron-astrocyte cocultures

In the adult CNS, axons fail to regenerate after injury. Among the cell interactions that lead to this failure are those developed with astrocytes. In an effort to elucidate the mechanisms underlying these negative interactions, we have used astrocytes treated with antisense glial fibrillary acidic protein (GFAP) mRNA to inhibit the formation of gliofilaments, indispensable for the astroglial morphological response to injury, and have studied their permissivity for neuritic outgrowth. In a neuron-astrocyte coculture, a mechanical lesion led to hypertrophy of astrocytes neighboring the lesion. Neuronal cell bodies and neurites were absent both from the area of lesion and from its surroundings. Reactive astrocytes appeared, therefore, to be a nonpermissive substrate. Transfection that used antisense GFAP mRNA blocked astroglial morphological changes and was characterized by both a persistence of neuronal cell bodies in the vicinity of the lesion site and a growth of neurites into the same region. These morphological differences were associated with a 46% decrease in the GFAP translation capacity and a 50% increase in the concentration of GAP-43 in the treated cultures. Neurons were associated mainly with an extracellular laminin network, which was predominant at the lesion site in treated cocultures. In contrast, those astrocytes highly laminin-immunoreactive appeared to be a nonpermissive substrate for neurons. These results show that inhibition in GFAP synthesis, leading to a reduction of astroglial hypertrophy, relieves the blockade of neuritic outgrowth that normally is observed after a lesion. The mechanisms may involve changes in the secretion of extracellular matrix molecules by astrocytes.     

Reactive astroglia-neuron relationships in the human cerebellar cortex: a quantitative, morphological and immunocytochemical study in Creutzfeldt-Jakob disease

In order to investigate the role of neuron-glia interactions in the response of astroglial to a non-invasive cerebellar cortex injury, we have used two cases of the ataxic form of Creutzfeldt-Jakob disease (CJD) with distinct neuronal loss and diffuse astrogliosis. The quantitative study showed no changes in cell density of either Purkinje or Bergmann glial cells in CJ-1, whereas in the more affected CJ-2 a loss of Purkinje cells and an increase of Bergmann glial cells was found. The granular layer in both CJD cases showed a similar loss of granule cells (about 60%) in parallel with the significant increase in GFAP+ reactive astrocytes. GFAP immunostaining revealed greater reactivity of Bergmann glia in CJ-2 than in CJ-1, as indicated by the thicker glial processes and the higher optical density. Granular layer reactive astrocytes were regularly spaced. In both CJD cases there was strict preservation of the spatial arrangement of all astroglial subtypes--Fa?anas cells, Bergmann glia and granular layer astrocytes. Reactive Fa?anas and Bergmann glial cells and microglia/macrophages expressed vimentin, while only a few vimentin+ reactive astrocytes were detected in the granular layer. Karyometric analysis showed that the increase in nuclear volume in reactive astroglia was directly related with the level of glial hypertrophy. The number of nucleoli per nuclear section was constant in astroglial cells of human controls and CJD, suggesting an absence of polyploidy in reactive astroglia. Ultrastructural analysis revealed junctional complexes formed by the association of macula adherens and gap junctions. In the molecular layer numerous vacant dendritic spines were ensheathed by lamellar processes of reactive Bergmann glia. Our results suggest that quantitative (neuron/astroglia ratio) and qualitative changes in the interaction of neurons with their region-specific astroglial partners play a central role in the astroglial response pattern to the pathogenic agent of CJD.     

A culture model of reactive astrocytes: increased nerve growth factor synthesis and reexpression of cytokine responsiveness

Reactive gliosis, which occurs in response to damage to the central nervous system, has been recognized for years but is not yet understood. We describe here a tissue culture model of reactive astrocytes used to characterize their properties. Cultures are prepared 1 week following 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra and compared with astrocytes cultured from normal adult rats or rats injected with saline only. Astrocytes from the 6-OHDA-lesioned side contained elevated levels of glial fibrillary acidic protein (GFAP) and GFAP mRNA and were intensely immunoreactive for GFAP, vimentin, and two epitopes that in vivo are found only on reactive astrocytes. The basal content of nerve growth factor (NGF) mRNA and NGF in astrocytes from 6-OHDA-lesioned rats was significantly higher relative to control astrocytes. Two inflammatory cytokines, interleukin-1beta and interferon-gamma, increased synthesis of NGF up to 20-fold in the reactive cells, whereas there was no response in the normal adult astrocytes. Astrocytes from postnatal day 2 rats shared many of the properties of the reactive adult astrocytes. These cultures offer the possibility to characterize the cellular and molecular properties of reactive astrocytes and to determine the factors responsible for activation of astrocytes.     

Inappropriate apoptosis of salivary and lacrimal gland epithelium of immunodeficient NOD-scid mice

Reactive gliosis, which occurs in response to any damage or disturbance to the central nervous system, has been recognized for many years, but is still not completely understood. The hallmark is the increased expression of glial fibrillary acidic protein (GFAP), yet studies in GFAP knockout mice suggest that GFAP may not be required for an astrocyte to become hypertrophic. In this review, we describe a series of tissue culture models that have been established in order to address: 1) the biochemical phenotype of reactive astrocytes; 2) the factor and/or cell responsible for induction of gliosis; 3) the mechanisms by which one might block the induction. These models range from cultures of astrocytes, both neonatal and adult, to co-cultures of astrocytes with either neurons or microglia, to organ cultures. None is ideal: each addresses a different set of questions, but taken together, they are beginning to provide useful information which should allow a better understanding of the plasticity response of astrocytes to brain injury.     

Intracerebral injection of interferon-gamma inhibits the astrocyte proliferation following the brain injury in the 6-day-old rat

The present study examines the influence of interferon-gamma (IFN-gamma) on the astrocyte proliferation in the rat brain injured within the early period of postnatal development. Six-day-old male rats received a lesion in the left cerebral hemisphere and a single injection of recombinant rat IFN-gamma into the lesion cavity. One or 2 days after the injury the rats were injected with 3H-thymidine. Brain sections were immunostained for glial fibrillary acidic protein (GFAP), subjected to autoradiography, and examined microscopically to record proliferating GFAP-immunopositive astrocytes labeled with 3H-thymidine. In the IFN-gamma-injected rats, a statistically significant decrease in the intensity of reactive astrocyte proliferation was revealed. On day 1 after injury the intensity of astrocyte proliferation showed dose-dependent changes. Relations between the astrocyte reactivity and multiple factors existing in the injured and IFN-gamma-injected brain are discussed. The results represent the first in vivo evidence of a dose-dependent action of IFN-gamma on the astrocyte proliferation in response to injury.     

Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds

Brain injury induces reactive gliosis, characterized by increased expression of glial fibrillary acidic protein (GFAP), astrocyte hypertrophy, and hyperplasia of astrocytes and microglia. One hypothesis tested in this study was whether ganglioside GD3+ glial precursor cells would contribute to macroglial proliferation following injury. Adult rats received a cortical stab wound. Proliferating cells were identified by immunostaining for proliferating cell nuclear antigen (PCNA) and by [3H]-thymidine autoradiography, and cell phenotypes by immunocytochemical staining for GD3, GFAP, ED1 (for reactive microglia) and for Bandeiraea Simplicifolia isolectin-B4 binding (all microglia). Animals were labeled with thymidine at 1,2,3, and 4 days postlesion (dpl) and sacrificed at various times thereafter. Proliferating cells of each phenotype were quantified. A dramatic upregulation of GD3 on ramified microglia was seen in the ipsilateral hemisphere by 2 dpl. Proliferating cells consisted of microglia and fewer astrocytes. Microglia proliferated maximally at 2-3 dpl and one third to one half were GD3+. Astrocytes proliferated maximally at 3-4 dpl, and some were also GD3+. Both ramified and ameboid forms of microglia proliferated and by 4 dpl all GD3+ microglia were ED1+ and vice versa. In the contralateral cortex microglia expressed neither GD3 nor ED1. Thus they acquired these antigens when activated. Neither microglia nor astrocytes that were thymidine-labeled at 2, 3, or 4 dpl changed in number in subsequent days. Most thymidine+ astrocytes were large GFAP+ reactive cells that clearly arose from pre-existing astrocytes, not from GD3+ glial precursors. In this model of injury microglia proliferate earlier and to a much greater extent than astrocytes, they can divide when in ramified form, and GD3 is up-regulated in most reactive microglia and in a subset of reactive astrocytes. We also conclude that microglial proliferation precedes proliferation of invading blood-borne macrophages.     

GFAP-deficient astrocytes are capable of stellation in vitro when cocultured with neurons and exhibit a reduced amount of intermediate filaments and an increased cell saturation density

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Pekny et al. (1995) EMBO J. 14, 1590-1598]. Astrocytes in culture, similar to reactive astrocytes in vivo, express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell density in vitro. GFAP-/- astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP-/- astrocytes compared to that in GFAP+/+ astrocytes. GFAP-/- astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP-/- astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.     

Temporal characterization of microglia, IL-1 beta-like immunoreactivity and astrocytes in the dentate gyrus of hippocampal organotypic slice cultures

These studies demonstrate that murine hippocampal slice cultures possess neural-immune elements that show responses parallel to comparable in vivo models of neural-immune activation. Using immunocytochemical techniques, this study characterized the phenotypes of specific glial elements and the expression of the cytokine, interleukin-1 (IL-1 beta), in the hippocampal dentate gyrus over a period of 10 days in vitro (DIV). Preparation of organotypic slice cultures of neonatal mouse hippocampus produced cellular damage including axotomy of afferent fibers within the molecular layer of the dentate gyrus. This form of lesion-induced injury caused activation of neural-immune elements in the slice cultures. Staining with the microglial specific biotinylated Griffonia simplicifolia B4-isolectin revealed reactive microglia were most prevalent at 2 DIV and decreased in number from 4 to 10 DIV, whereas the initial population of resting microglia at 2 DIV increased approximately four-fold from 4 to 10 DIV. The presence of a round IL-1 beta-like immunophenotype closely paralleled the temporal and spatial distribution of the reactive form of microglia observed in the dentate gyrus. In addition, between 4 and 10 DIV, some IL-1 beta-like immunoreactive cells exhibited a stellate-like morphology with numerous branching processes, similar to resting microglia. At 2 DIV astrocytes showed minimal labeling with antibodies directed against glial fibrillary acidic protein (GFAP), while from 4 to 10 DIV, a dramatic hypertrophic astrocytic response occurred, resulting in a gliotic scar forming over the entire dentate gyrus. We conclude that neural-immune activation in the hippocampal organotypic slice culture preparation closely parallels similar responses observed in vivo and thus slice cultures represent an excellent model for further studies of neural-immune interactions resulting from lesion-induced injury in the central nervous system.     

Glial fibrillary acidic protein expression but no glial demarcation follows the lesion in the molecular layer of cerebellum

The present study investigates the reactive gliosis following a simple stab wound lesion to a brain area in which a characteristic astroglial architecture exists, i.e., the Bergmann-glia in the molecular layer of cerebellum. While in mammalian brain the Bergmann-glia contains glial fibrillary acidic protein (GFAP), in the avian Bergmann-glia, the cytoskeletal protein is vimentin, which is characteristic for immature astroglia in mammals. The operations were performed on chickens and rats under deep anaesthesia, using a sterile disposable needle. After a 1-week survival period, the animals were overdosed with ether and perfused transcardially with 4% buffered paraformaldehyde. Free-floating sections cut with a vibration microtome were processed for immunohistochemistry against GFAP and vimentin. GFAP immunopositivity of Bergmann-glia appeared in chicken and increased in rat in the lesioned area but the lesion was not surrounded by typical astrocytes and no demarcation was formed in the molecular layer, in contrast to the usual appearance of reactive gliosis, which was observed in the granular layer and in the white matter in both species. Vimentin immunopositivity of the Bergmann-glia also increased around the lesion in both species. The results suggest that a highly developed glial architecture fails to re-arrange into a demarcating scar, which offers an interesting model system to study the importance of glial demarcation. The observations also support that the resident glia is the main component of the glial reaction, and prove the capability of avian Bergmann-glia to express GFAP.     

Time course of 5-HT2A receptor occupancy in the human brain after a single oral dose of the putative antipsychotic drug MDL 100,907 measured by positron emission tomography

Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-d-old rat pups exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through the mother's milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocampus and cerebellum. A quantitative analysis of reactive astrocytes was performed by using GFAP and S-100 protein as specific markers. The study showed a significant increase in their number, size, number of processes, and density of immunostaining in 2,4-D-exposed animals. Exposure to 2,4-dichlorophenoxyacetic acid on the first days of life modifies the astroglial cytoarchitecture in parallel to previously described neuronal changes.     

Region-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor

Brains from human neurofibromatosis type 1 (NF1) patients show increased expression of glial fibrillary acidic protein (GFAP), consistent with activation of astrocytes (M.L. Nordlund, T.A. Rizvi, C.I. Brannan, N. Ratner, Neurofibromin expression and astrogliosis in neurofibromatosis (type 1) brains, J. Neuropathol. Exp. Neurology 54 (1995) 588-600). We analyzed brains from transgenic mice in which the Nf1 gene was targeted by homologous recombination. We show here that, in all heterozygous mice analyzed, there are increased numbers of astrocytes expressing high levels of GFAP in medial regions of the periaqueductal gray and in the nucleus accumbens. More subtle, but significant, changes in the number of GFAP positive astrocytes were observed in the hippocampus in 60% of mutant mice analyzed. Astrocytes with elevated GFAP were present at 1 month, 2 months, 6 months and 12 months after birth. Most brain regions, including the cerebellum, basal ganglia, cerebral cortex, hypothalamus, thalamus, cortical amygdaloid area, and white matter tracts did not show any gliotic changes. No evidence of degenerating neurons was found using de Olmos' cupric silver stain. We conclude that Nf1/nf1 mice provide a model to study astrogliosis associated with neurofibromatosis type 1.     

Alterations in temporal/spatial distribution of GFAP- and vimentin-positive astrocytes after spinal cord contusion with the New York University spinal cord injury device

Astrocytes become reactive as a result of various types of lesions and upregulate 2 intermediate filaments, glial fibrillary acidic protein (GFAP), and the developmentally regulated protein vimentin. Young female Sprague-Dawley rats were subjected to a spinal cord contusion at segment T10 using the New York University injury device. Animals were killed at 1, 2, 7, 14, and 30 days postinjury. Horizontal spinal cord sections spanning segments T7-T13 were assessed with antibodies to both intermediate filament proteins. The number of gray matter GFAP-positive astrocytes increased by 2 days postinjury, with segments adjacent (proximal) to the injury site showing greater responses than areas several segments away (distal). By 30 days following injury, astroglial cell numbers returned to normal levels. Vimentin-positive astrocytes also showed a graded proximal/distal response by 2 days following injury. Proximal regions remained significantly higher at 30 days following injury than control animals. Rostral/caudal changes were also evident, with regions caudal to the injury showing significantly higher numbers of vimentin positive astrocytes than those rostral, indicating that gray matter areas caudal to spinal cord injury may undergo more stress following spinal cord injury.     

Effects of prenatal gamma-irradiation on the astrocyte proliferation in response to injury in the brain of 6-day-old rat

Pregnant Wistar rats were exposed to a single 1.0 Gy dose of gamma rays on gestational days 13, 15, 17 or 19 (E13, E15, E17 and E19, respectively). A mechanical injury was made in the cerebral hemisphere of their 6 day-old male offsprings. The injured rats were injected with [3H]thymidine on day 1 or 2 after injury and killed 4 h after the injection. Brain sections were immunostained for glial fibrillary acidic protein (GFAP) or S-100beta protein, subjected to autoradiography and examined microscopically to record proliferating astrocytes. The intensity of astrocyte proliferation in response to injury showed a gradual decrease from the level maximal in brains irradiated on E13 to minimal in those irradiated on E19. The changes were regarded as being related to the stage of prenatal development when irradiation of the brain was performed.     

Accuracy of automated finger blood pressure devices

Substance P and glial fibrillary acidic protein (GFAP) immunohistochemistry was applied to the medulla of neonatal infants who died of sudden infant death syndrome (SIDS). A quantitative analysis of cells demonstrating immunoreactivity to GFAP and substance P in 15 neonatal SIDS cases revealed increased GFAP immunoreactivity in the reticular formation, the dorsal vagal nucleus, and the solitary nucleus and an increase in substance P immunoreactivity in the spinal trigeminal nucleus and the solitary nucleus as compared with that in age-matched controls. GFAP immunopositivity suggests astrogliosis which implies a pathologic insult to neurons in the area of astrogliosis. The failure of neurons in these sites to show enhanced substance P immunopositivity may indirectly indicate altered neurons. Further study of prenatal events may be of importance in clarifying the pathogenesis of neonatal SIDS.     

Deprenyl induces GFAP immunoreactivity in the intact and injured dopaminergic nigrostriatal system but fails to counteract axotomy-induced degenerative changes

There is increasing evidence of a trophic-like mechanism for some effects ascribed to deprenyl therapy in the central nervous system. For that, we studied the effect of chronic treatment with deprenyl in an animal model of Parkinson's disease induced by unilateral knife transection of the medial forebrain bundle (MFB) in adult rats. The experimental conditions included a 3-week pretreatment with deprenyl before stereotaxic transection of the MFB. Following surgery, deprenyl treatment was maintained for 3 weeks. Neurochemical and immunohistochemical procedures were used to study the dopaminergic system and reactive astrocytes in the nigrostriatal system. Deprenyl treatment failed to counteract the axotomy-induced degenerative changes of the nigrostriatal dopaminergic system. However, it was effective in increasing the density of reactive astrocytes in terms of glial fibrillary acidic protein (GFAP) immunoreactivity in the intact contralateral substantia nigra and also in further enhancing the axotomy-induced increase of GFAP immunolabeled astrocytes in the lesioned substantia nigra. This deprenyl-induced effect on GFAP immunoreactivity was confined to substantia nigra without effect in striatum. In addition, we found a medial to lateral gradient decrease in the distribution pattern of GFAP immunolabeled astrocytes. Axotomy increased the number of reactive astrocytes in either striatal area examined, but yet the preferential distribution pattern of reactive astrocytes in striatum was still evident.     

Distribution of glial fibrillary acidic protein and vimentin-immunopositive elements in the developing chicken brain from hatch to adulthood

The present study describes the distribution of glial fibrillary acidic protein (GFAP) and vimentin-immunopositive structures in the brain of the domestic chicken (Gallus domesticus) from hatching to maturity. The telencephalon is penetrated by a vimentin-immunopositive radial fibre system, representing a modified form of radial glia, in day-old chicks. Numerous fibres of this system persist until adulthood, mainly in the lobus parolfactorius, lamina medullaris dorsalis and lamina frontalis superior. GFAP immunoreactivity also appears in the course of development in these fibres. The distribution of GFAP-immunopositive astrocytes in the post-hatch telencephalon is like that found in adult chicken, except for the ectostriatum, in which an adult-like GFAP-immunostaining only develops during week three. This delay may be associated with a relatively slow maturation of this visual centre. In the diencephalon and in the mesencephalic tegmentum of day-old chicks GFAP-immunopositive astrocytes are confined to the border zone of several nuclei. In these areas as well as in the pons most GFAP positive astrocytes only appear gradually during the first two post-hatch weeks, although radial fibres occur only sparsely at hatch. Summarizing these results, a gradual replacement of radial fibres by astrocytes, typical of mammals, cannot be found in chicken. In the nucleus laminaris we observed a characteristic palisade of non-ependymal glia, reactive to GFAP but not to vimentin, which almost completely disappears by adulthood. We suggest that this glial system is instrumental in the development of the dendritic organisation of this nucleus. The optic tectum displays a dense array of GFAP-immunopositive radial glia at hatching, similar in this to the situation found in reptiles. However, in the tectum of reptiles this radial glia persists for the lifetime, whereas in the chick it disappears from the superficial tectal layers. This phenomenon may reflect the fact that there is no replacement of tectal cells or regeneration of retinotectal pathways in the chicken. In the early stage, the large cerebral tracts were found to contain dense accumulations of GFAP-positive cells, with peculiarly long outgrowths accompanying nerve fibres. No vimentin-immunopositivity was found in these glial elements; however vimentin was present in the glia situated at the optic chiasm, the anterior commissure and at other decussations. These structures, as well as the raphe, displayed the most intense vimentin-immunopositivity in the post-hatch chicken. This characteristic glial population may represent glial elements that have been reported to regulate fibre-crossing at the midline.     

The relation between rhinitis and allergic asthma. The action of antihistaminics on bronchial hyperreactivity

The intermediate filament nestin is highly expressed in multipotential stem cells of the developing central nervous system (CNS). During neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g. neurofilaments and glial fibrillary acidic protein (GFAP). In this study, we demonstrate that nestin expression is re-induced in reactive astrocytes in the lesioned adult brain. Following ischaemic and mechanical lesioning, a strong and sustained expression of nestin was noted in GFAP-positive cells surrounding the lesion site. Lesion experiments in transgenic mice carrying the lacZ gene under control of regulatory sequences from the nestin gene suggested that the upregulation of nestin in reactive astrocytes is mediated via the same sequences that control nestin expression during CNS development. These observations and recent data on the co-expression of glial and neuronal marker antigens in reactive astrocytes point to a close relationship between proliferating astrocytes and neuroepithelial precursor cells.     

Effects of axonal injury on astrocyte proliferation and morphology in vitro: implications for astrogliosis

Mechanisms inducing gliosis following injury in the central nervous sy stem are poorly understood. We evaluated the effect of axonal injury on astrocyte and Schwann cell proliferation and morphology in vitro. Purified rat dorsal root ganglion neurons grown on monolayers of rat neonatal cortical astrocytes (N-ASneonatal cultures) or sciatic nerve-derived Schwann cells (N-SC cultures) were mechanically injured. Non-injured cultures served as controls. Cell proliferation near lesions was monitored by autoradiography 1,2,4, and 8 days postinjury. Axonal injury caused a significant transient increase in astrocyte proliferation immediately proximal and distal to the lesion. The lesion did not induce marked changes in the intensity of glial fibrillary acidic protein (GFAP) immunoreactivity. However, processes from GFAP-positive cells usually arranged in random fashion in noninjured cultures were aligned perpendicularly to the cut distal to lesions. Ultrastructural analysis in lesioned N-ASneonatal cultures indicated that proximal to the lesion filament-filled astrocytes were intermingled with axons. Distal to the lesion astrocyte processes formed layers, between which an increased amount of collagen-like material appeared with time postlesion. Axons distal to the lesion degenerated by 2 days, coinciding with the early disappearance of neurofilament immunoreactivity. In noninjured and proximally in injured N-SC cultures, Schwann cells extended processes, engulfing some axons. Distal to the lesion, Schwann cells appeared more rounded and neurites remained until 4 days postinjury. Media conditioned by injured or non-injured N-ASneonatal cultures did not affect neuron-induced Schwann cell proliferation. These findings demonstrate that axonal injury and degeneration cause a transient increase in astrocyte proliferation and induce morphological changes in astrocytes consistent with the onset of gliosis.