Age-dependent changes at the blood-brain barrier. A Comparative structural and functional study in young adult and middle aged rats.

Decreased beta-amyloid clearance in Alzheimer's disease and increased blood-brain barrier permeability in aged subjects have been reported in several articles. However, morphological and functional characterization of blood-brain barrier and its membrane transporter activity have not been described in physiological aging yet. The aim of our study was to explore the structural changes in the brain microvessels and possible functional alterations of P-glycoprotein at the blood-brain barrier with aging. Our approach included MR imaging for anatomical orientation in middle aged rats, electronmicroscopy and immunohistochemistry to analyse the alterations at cellular level, dual or triple-probe microdialysis and SPECT to test P-glycoprotein functionality in young and middle aged rats. Our results indicate that the thickness of basal lamina increases, the number of tight junctions decreases and the size of astrocyte endfeet extends with advanced age. On the basis of microdialysis and SPECT results the P-gp function is reduced in old rats. With our multiparametric approach a complex regulation can be suggested which includes elements leading to increased permeability of blood-brain barrier by enhanced paracellular and transcellular transport, and factors working against it. To verify the role of P-gp pumps in brain aging further studies are warranted.

Probing nano-organization of astroglia with multi-color super-resolution microscopy.

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100ß and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc.

Application of high-intensity focused ultrasound to the study of mild traumatic brain injury.

Though intrinsically of much higher frequency than open-field blast overpressures, high-intensity focused ultrasound (HIFU) pulse trains can be frequency modulated to produce a radiation pressure having a similar form. In this study, 1.5-MHz HIFU pulse trains of 1-ms duration were applied to intact skulls of mice in vivo and resulted in blood-brain barrier disruption and immune responses (astrocyte reactivity and microglial activation). Analyses of variance indicated that 24 h after HIFU exposure, staining density for glial fibrillary acidic protein was elevated in the parietal and temporal regions of the cerebral cortex, corpus callosum and hippocampus, and staining density for the microglial marker, ionized calcium binding adaptor molecule, was elevated 2 and 24 h after exposure in the corpus callosum and hippocampus (all statistical test results, p < 0.05). HIFU shows promise for the study of some bio-effect aspects of blast-related, non-impact mild traumatic brain injuries in animals.

Three-dimensional reconstruction of the pharyngeal tonsil innervation pattern in sheep.

The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.

Attenuation of corpus callosum axon myelination and remyelination in the absence of circulating sex hormones.

Sex differences in the structure and organization of the corpus callosum (CC) can be attributed to genetic, hormonal or environmental effects, or a combination of these factors. To address the role of gonadal hormones on axon myelination, functional axon conduction and immunohistochemistry analysis of the CC in intact, gonadectomized and hormone-replaced gonadectomized animals were used. These groups were subjected to cuprizone diet-induced demyelination followed by remyelination. The myelinated component of callosal compound action potential was significantly decreased in ovariectomized and castrated animals under normal myelinating condition. Compared to gonadally intact cohorts, both gonadectomized groups displayed more severe demyelination and inhibited remyelination. Castration in males was more deleterious than ovariectomy in females. Callosal conduction in estradiol-supplemented ovariectomized females was significantly increased during normal myelination, less attenuated during demyelination, and increased beyond placebo-treated ovariectomized or intact female levels during remyelination. In castrated males, the non-aromatizing steroid dihydrotestosterone was less efficient than testosterone and estradiol in restoring normal myelination/axon conduction and remyelination to levels of intact males. Furthermore, in both sexes, estradiol supplementation in gonadectomized groups increased the number of oligodendrocytes. These studies suggest an essential role of estradiol to promote efficient CC myelination and axon conduction in both sexes.

Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer's disease.

Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimer's disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.

Apolipoprotein D expression in human brain reactive astrocytes.

Astrocytosis is a hallmark of damage that frequently occurs during aging in human brain. Astrocytes proliferate in elderly subjects, becoming hypertrophic and highly immunoreactive for glial fibrillary acidic protein (GFAP). These cells are one type that actively responds in the repair and reorganization of damage to the neural parenchyma and are a source of several peptides and growth factors. One of these biomolecules is apolipoprotein D (apo D), a member of the lipocalin family implicated in the transport of small hydrophobic molecules. Although the role of apo D is unknown, increments in brain apo D expression have been observed in association with aging and with some types of neuropathology. We have found an overexpression of apo D mRNA in reactive astrocytes by in situ hybridization in combination with immunohistochemistry for apo D in normal aged human brains. The number of double-labeled cells varied according to the cerebral area and the gliosis grade. The possible significance of this increased synthesis of apo D in reactive astrocytes is discussed in relation to the role of apo D in aging and in glial function.

Different glial response to methamphetamine- and methylenedioxymethamphetamine-induced neurotoxicity.

The consequences of the neurotoxic insult induced by 3,4-methylenedioxymethamphetamine (MDMA, an amphetamine derivative with specific action on the serotonergic system) were compared with those of methamphetamine (a derivative with specific action on dopaminergic system) in rats. Both drugs induced a very similar loss of body weight, especially evident 24 h after treatment. Their hyperthermic profile was also very similar and was dependent on ambient temperature, corroborating the thermo-dysregulatory effect of both substances. Methamphetamine (four injections of 10 mg kg(-1) s.c. at 2-h intervals) induced the loss of dopaminergic (35%) but not of serotonergic, terminals in the rat striatum and, simultaneously, a significant increase in striatal peripheral-type benzodiazepine receptor density, pointing to a glial reaction. Evidence for this drug-induced astrogliosis was the increased heat shock protein 27 (HSP27) expression in striatum, cortex and hippocampus. MDMA (20 mg kg(-1) s.c. b.i.d. for 4 days) induced a similar dopaminergic lesion in the striatum 3 days post-treatment, which reversed 4 days later. An important neurotoxic effect on serotonergic terminals was also observed in the cortex, striatum and hippocampus 3 days post-treatment, which partially reversed 4 days later in the striatum and hippocampus. No microglial activation was noticeable at either 3 or 7 days after MDMA treatment. This lack of effect on microglial cells was assessed by [(3)H]PK 11195 binding and OX-6 immunostaining, which were unchanged in the striatum and cortex after MDMA treatment. A non-significant tendency to increase was noted in the hippocampus 3 days after MDMA treatment. Furthermore, in MDMA-treated rats, neither HSP27 expression nor an increase in HSP27 immunoreactivity were detected. This result, together with the lack of increase in glial fibrilliary acidic protein (GFAP) immunoreactivity, indicate no astroglial activation at either 3 or 7 days post-treatment. Without microglial activation, an inflammatory process would not accompany the lesion induced by MDMA. The differences in glial activation between methamphetamine and MDMA observed in the present study could have implications for the prognosis of the injury induced by these drugs.

Clinorotation-induced weightlessness influences the cytoskeleton of glial cells in culture.

During and after spaceflight astronauts experience neurophysiological alterations. To investigate if the impairment observed might be traced back to cytomorphology, we undertook a ground based research using a random positioning machine (clinostat) as a simulation method for microgravity. The outcome of the study was represented by cytoskeletal changes occurring in cultured glial cells (C(6) line) after 15 min, 30 min, 1 h, 20 h and 32 h under simulated microgravity. Glia is fundamental for brain function and it is essential for the normal health of the entire nervous system. Our data showed that after 30 min under simulated microgravity the cytoskeleton was damaged: microfilaments (F-actin) and intermediate filaments (Vimentin, Glial Fibrillary Acidic Proteins GFAP) were highly disorganised, microtubules (alpha-tubulin) lost their radial array, the overall cellular shape was deteriorated, and the nuclei showed altered chromatin condensations and DNA fragmentation. This feature got less dramatic after 20 h of simulated microgravity when glial cells appeared to reorganise their cytoskeleton and mitotic figures were present. The research was carried out by immunohistochemistry using antibodies to alpha-tubulin, vimentin and GFAP, and cytochemical labelling of F-actin (Phalloidin-TRIC). The nuclei were stained with propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed at the conventional and/or the confocal laser scanning microscope. Samples were also observed at the scanning electron microscope (SEM). Our data showed that in weightlessness alterations occur already visible at the scale of the single cell; if this may lead to the neurophysiological problems observed in flight is yet to be established.

The exact expression of glial fibrillary acidic protein (GFAP) in trigeminal ganglion and dental pulp.

The expression in various cell types of peripheral tissues of glial fibrillary acidic protein (GFAP), first discovered as an intermediate filament specific for astrocytes, remains controversial owing to numerous reports of a wide distribution for GFAP-immunoreactivity in various cells. The present study employed immunohistochemistry to investigate the precise expression of GFAP in the dental pulp and trigeminal ganglion of adult rats and wild-type mice as well as GFAP-knockout mice. The exhibition of GFAP-immunoreactivity in the trigeminal ganglion was further examined by a reverse transcription polymerase chain reaction (RT-PCR) technique, and in situ hybridization histochemistry using a specific cRNA probe prepared by us. The immunoreaction for GFAP was recognizable in the axons, Schwann cells, and the fibroblasts in the dental pulp of rats and wild-type littermate mice. However, mice with null mutations in the GFAP gene remained immunoreactive for GFAP in all these locations. Intense GFAP-immunoreactivity was found in a small number of satellite cells in the trigeminal ganglion in all animals examined in this study. RT-PCR analysis demonstrated bands for the GFAP gene corresponding to the length expected from the primer design in the samples of trigeminal ganglion and dental pulp. In situ hybridization histochemistry also showed intense signals for GFAP mRNA in some satellite cells of the trigeminal ganglion, but never in the neurons. These data suggest that the GFAP-immunoreactive molecules in the pulpal axons and fibroblasts react non-specifically with the polyclonal antibody and are probably a closely related type of intermediate filament.

Ultrastructural and immunohistochemical findings in five patients with vitreomacular traction syndrome.

OBJECTIVE: To evaluate pathologic features of vitreomacular traction syndrome (VMT). METHODS: Preretinal membranes removed from five patients during vitreous surgery for VMT syndrome were evaluated by electron microscopy (n = 4) and immunohistochemistry (n = 1). RESULTS: Electron microscopic examination revealed large segments of internal limiting lamina (ILL) in three of the four cases. Other extracellular features included two types of abnormal collagen fibrils, determined to be type I and fibrous long-spacing collagen. The myofibrocyte was the predominant cell type in all cases. Collagen types I, II, and III, as well as glial fibrillary acidic protein and vimentin, were identified by immunohistochemistry. CONCLUSIONS: Epiretinal membranes in eyes with VMT syndrome adhered tightly to the ILL with abnormal collagen and contractile elements that included myofibrocytes.

Electron immunocytochemical analysis of posterior hyaloid associated with diabetic macular edema.

BACKGROUND: Tangential traction in the macula from a thickened posterior hyaloid of the vitreous has been implicated as a cause of diffuse diabetic macular edema. Vitrectomy with peeling of the posterior hyaloid has been shown to reduce retinovascular leakage and improve vision in select patients. We report a clinicopathologic correlation using electron microscopy and electron immunocytochemistry to characterize the membrane infiltrating the posterior hyaloid in two such patients. METHODS: Two patients presented with vision loss associated with diffuse diabetic macular edema and an attached, thickened, and taut posterior hyaloid. The patients underwent vitrectomy with peeling of the posterior hyaloid. The premacular posterior hyaloid specimens then were analyzed by electron microscopy with immunocytochemical staining for cytokeratin and glial fibrillary acidic protein. RESULTS: Both posterior hyaloid specimens contained collagen and a large cellular component. Immunogold labeling showed cells positive for glial fibrillary acidic protein or cytokeratin. With double labeling, no cells expressed both proteins simultaneously. Clinically, both patients had vision improvement and macular edema resolution after surgery. CONCLUSIONS: The thickened, taut posterior hyaloid observed in our patients with diabetic macular edema contained cells of glial and epithelial origin. This cellular infiltration may contribute to abnormal vitreomacular adherence and could play a role in the pathogenesis of macular edema in some patients with diabetes.

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.

Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study.

Experimentally induced demyelination due to the direct injection of gliotoxic agents has provided powerful models for studying the biology of remyelination. For the most part, these models have involved injection into white matter tracts of the spinal cord. However, the spinal cord has a number of limitations, such as the size of lesions that it is possible to make and its unsuitability for long-term direct cannulation for the delivery of putative remyelination-enhancing agents. In this study, we describe the natural history of three new models of demyelination/remyelination based on the stereotaxic injection of three gliotoxins: lysolecithin, ethidium bromide, and a combination of anti-galactocerebroside antibody and complement (GalC-ab/comp) into the caudal cerebellar peduncle of adult rats. All three agents produced large areas of demyelination with minimal axonal damage, which undergo extensive remyelination. Ethidium bromide- and GalC-ab/comp-induced lesions remyelinated more slowly than those induced by lysolecithin. The contribution to the remyelination of the lesion by Schwann cells reflects the degree of astrocyte damage incurred within the demyelinated area and is greatest for ethidium bromide-induced demyelination. These new models not only provide further insights into the mechanisms of CNS remyelination but also provide a valuable new resource for addressing a series of key issues relevant to current efforts to promote CNS remyelination either by the enhancement of intrinsic processes or by the transplantation of myelinogenic cells.

[Central neurocytoma]. / Neurocitoma central.

INTRODUCTION: A central neurocytoma (CN) is a rare tumor, of neuronal origin, well-differentiated and found intraventricularly. It mainly affects young adults. Firm diagnosis is made on immunohistochemical (IHQ) and ultrastructural studies, since on optic microscopy it is similar in appearance to an oligodendroglioma or to an ependymoma. PATIENTS AND METHODS: We studied 4 cases, three after surgical resection and one on autopsy. The average age was 29, ranging from 3 to 63. Both sexes were equally affected. In all cases IHQ techniques were used (GFAP, neurofilament, synaptophysin and specific neuronal enolase) and they were studied by electron microscopy. RESULTS: IHQ was negative for GFAP and neurofilament, but intensely positive for synaptophysin and specific neuronal enolase. On ultrastructural study there were few neurofilaments, microtubules and dense central granules typical of neural differentiation. CONCLUSIONS: The findings in our cases lead to diagnosis of NC and confirm that this tumor is a distinct clinicopathological entity.

An immunocytochemical study of isolated human retinal Müller cells in culture.

BACKGROUND: We report a modified method for the isolation and propagation of adult human Müller cells in culture. METHODS: The retina of postmortem human eyes was mechanically dissociated and cultured. Using immunocytochemical techniques, these cells were stained with monoclonal antibodies specific for Müller cells, glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS) and keratin. Transmission electron microscopy (TEM) was also performed. RESULTS: The dissociated and cultured cells expressed vimentin and GS, but not GFAP. At least 85% of these cells stained with a Müller cell-specific monoclonal antibody. Using TEM, flat cells containing 13-nm intermediate filaments and glycogen were identified. CONCLUSION: Human retinal Müller cells can be isolated and propagated in culture. Purified cell cultures are required for controlled studies of the normal physiology and pathologic responses of Müller cells.

A monoclonal antibody to astrocytes, subepithelial fibroblasts of small intestinal villi and interstitial cells of the myenteric plexus layer.

A monoclonal antibody was developed, using cultured subepithelial fibroblasts of rat duodenal villi as the antigenic material, by in vitro immunization. Hybridomas were selected on cryosections of rat brain and small intestine using indirect immunofluorescence techniques. The monoclonal antibody, termed 8E1, was very useful to label GFAP-positive astrocytes in the adult rat brain, subepithelial fibroblasts of intestinal villi and Thuneberg's fibroblast-like interstitial cells associated with the myenteric plexus layer. These cells are known to be stellate in shape, forming a cellular network with extended cell processes that communicate via gap junctions. Immature astrocytes such as radial fibers were not immunolabeled with mAb-8E1. Immunoelectron microscopy revealed clustered gold labeling of 8E1 antigen on the intermediate filaments of cultured astrocytes and cultured subepithelial fibroblasts. This staining pattern was different from the continuous and linear gold labeling of GFAP and vimentin. MAb-8E1 did not immunoblot purified human brain GFAP nor bovine lens vimentin, and so was not neutralized by preabsorption with these molecules. Immunoblot analysis after SDS-PAGE showed that the antigen recognized by mAb-8E1 was a Mr 50K protein in the rat cerebra, and a Mr 56K protein in the cultured subepithelial fibroblasts. This study showed that astrocytes and some types of stellate cells share distinct antigenic properties in the components of their intermediate filaments.

The neuropathology of chromosome 17-linked dementia.

We recently described a family with chromosome 17-linked dementia, characterized clinically by disinhibition-dementia-parkinsonism-amyotrophy complex. We report now the neuropathology of 6 affected family members. This included semiquantitative scoring of neuronal loss, gliosis, and spongiosis and immunocytochemical and ultrastructural characterization of neuronal and glial inclusions. The changes consisted of circumscribed neuronal loss, gliosis, and spongiosis of limbic neocortical areas and frontal, temporal, and occipital association areas. Similar changes were present in subcortical nuclei, most severe in the substantia nigra, but also involved the ventral striatum and amygdala. The hippocampus was spared except for degeneration of the afferent perforant tract, secondary to entorhinal nerve cell loss. Argyrophilic neuronal inclusions, with a characteristic immunocytochemical profile, were found in brainstem nuclei, hypothalamus and basal ganglia. Ultrastructurally, in 3 patients these inclusions showed hitherto undescribed abnormally assembled filaments. Glial cytoplasmic inclusions were widespread in white matter structures. Immunocytochemistry failed to demonstrate the protease-resistant prion protein. The pathology appears to be unique, involving various cortical and subcortical structures, and is consistent with the clinical findings of Kluver-Bucy-like syndrome, parkinsonism, and frontal lobe dementia. For this entity we suggest the term "chromosome 17-linked dementia."

Neuroectodermal antigens persist in benign and malignant salivary gland tumor cultures.

OBJECTIVE: To determine whether a heterogeneous collection of salivary gland tumors shared common antigenic characteristics and growth patterns in tissue culture. DESIGN: Cell cultures were derived from benign and malignant salivary gland neoplasms, cultured conservatively, and serially analyzed for epithelial, myoepithelial, and neuroectodermal antigens. SUBJECTS: Nineteen samples reflecting the spectrum of salivary tumor pathologic characteristics were established in tissue culture. Most were derived from benign pleomorphic adenomas, and several were from carcinomas, including carcinoma ex pleomorphic adenoma, and mucoepidermoid and adenoid cystic carcinoma. RESULTS: All cultures were epithelial as determined by morphologic and antigenic examination, using antibodies for cytokeratin. The phenotype of cells derived from benign tumors, especially the pleomorphic adenomas, resembled those in the original neoplasm. Those from carcinomas were similar, with less differentiated characteristics. Manipulation of growth conditions altered the phenotypes shown in culture. Some cultures contained cells expressing vascular smooth-muscle actin and glial fibrillary acidic protein or nestin. CONCLUSIONS: This model cell system containing proliferative cells from several tumor types is consistent with a stem-cell theory of salivary gland tumor origin. Our data were not consistent with the bicellular or multicellular theory. We hypothesize a neuroectodermal origin for this group of apparently heterogeneous tumors. These cultured cells will be valuable for in-depth investigation of the loss of proliferation controls in benign and malignant tumors of the salivary gland.

Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.