Vimentin In Astrocytes Research Articles

Nerve Root Compression Increases Spinal Astrocytic Vimentin in Parallel With Sustained Pain and Endothelial Vimentin in Association With Spinal Vascular Reestablishment.

Temporal immunohistochemistry analysis of spinal cord tissue from a rat model of cervical radiculopathy. The goal was to measure spinal endothelial and astrocytic vimentin expression after a painful nerve root compression to define spinal cellular expression of vimentin in the context of pain. The intermediate filament, vimentin, is expressed in a variety of cell types in the spinal cord and is modulated in response to neural pathologies. Early after nerve root compression spinal astrocytes become activated and blood-spinal cord barrier (BSCB) breakdown occurs in parallel with development of pain-related behaviors; these spinal responses remain activated as does the presence of pain. In addition to vimentin, glial fibrillary acidic protein (GFAP) expression is a hallmark of astrocyte activation. In contrast, vascular endothelial cells down-regulate vimentin expression in parallel with vascular breakdown. It is not known whether spinal astrocytes and endothelial cells modulate their expression of vimentin in response to a painful neural injury. Mechanical hyperalgesia was measured and spinal cord tissue was harvested at days 1 and 7 after a unilateral nerve root compression in rats. Vimentin was coimmunolabeled with GFAP to label astrocytes and von Willebrand factor (VWF) for endothelial cells in the spinal cord on the side of injury. Spinal astrocytic vimentin increases by day 7 after nerve root compression, corresponding to when mechanical hyperalgesia is maintained. Spinal endothelial vimentin increases as early as day 1 after a painful compression and is even more robust at day 7. The delayed elevation in spinal astrocytic vimentin corresponding to sustained mechanical hyperalgesia supports its having a relationship with pain maintenance. Further, since BSCB integrity has been shown to be reestablished by day 7 after a painful compression, endothelial expressed vimentin may help to fortify spinal vasculature contributing to BSCB stability. N/A.

A Novel and Efficient Gene Transfer Strategy Reduces Glial Reactivity and Improves Neuronal Survival and Axonal Growth In Vitro

BackgroundThe lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi).Methods and FindingsIn this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model — scratched primary cultured astrocytes — Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi.ConclusionsLentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions.

Reactive changes in the adult rat superior colliculus after deafferentation

We have recently shown that optic nerve transection is followed by a delayed appearance (7-14 days after lesion) of putative guidance activities for retinal axons in the superior colliculus (SC) of adult rats. In the present study we analysed with immunohistological methods whether there is a correlation between localized reactive changes in the cellular compartment of the SC after deafferentation and the reappearance of putative guidance activities. The optic nerve of adult female Sprague-Dawley rats was transected. After 1 day to 1 year the contralateral SC was sectioned sagitally and examined with antibodies directed against Vimentin and glial fibrillary acidic protein (GFAP) to label astrocytes, myelin basic protein (MBP) to detect oligodendrocytes, OX-42 to stain microglia and SMI-31 to determine degeneration of afferent retinal ganglion cell axons. These stainings were performed in order to detect differences in the cellular composition of macro- and microglial cells between the anterior and posterior regions of the SC. All cell populations we examined in the SC showed reactive changes after optic nerve transection. Microglia activation occurred as soon as 2 days after optic nerve transection in the anterior part of the SC. After 4 days, a homogeneous distribution of activated microglia was seen in the whole SC. GFAP upregulation occurred without a specific regional distribution. Only Vimentin-expressing astrocytes appeared with a delay of 1-2 weeks after deafferentation and were strongly restricted to the afferent optic tract and the very anterior region of the SC. In conclusion, a temporo-spatial correlation between the re-expression of guidance activities and changes in the cellular compartment of the SC exists in the delayed re-expression of Vimentin in astrocytes and their strong spatial restriction. Activated microglia might be involved in triggering regional changes in an anterior-posterior graded fashion in the deafferented SC.

Fixation-dependent vimentin immunoreactivity of mono- and polyclonal antibodies in brain tissue of cattle, rabbits, rats and mice

The immunohistochemical staining of vimentin in paraffin-embedded sections from adult cattle, rabbit, rat and mouse brain fixed in different fixatives (formaldehyde, methacarn, ethanol) was examined using two monoclonal antibodies and a polyclonal antiserum. In non-trypsinized formaldehyde-fixed tissue sections both monoclonal antibodies and the polyclonal antibodies failed to stain vimentin. Following trypsinization of formaldehyde-fixed sections of the four species the meninges, endothelial cells of blood vessels, ependymal cells and the stroma of the choroid plexus were labelled by the monoclonal and polyclonal antibodies used. Astrocytes and Bergmann glial fibers in pretrypsinized formaldehyde-fixed sections from cattle, rabbit and rat brain, however, showed only weak staining. Fixation of cattle and rat brain in methacarn markedly improved the vimentin immunoreactivity of astrocytes and Bergmann glial fibers. The best fixative for the preservation of immunoreactive determinants of vimentin in astrocytes and Bergmann glial fibers in cattle, rabbit and rat brain was ethanol. In brain tissue from mice both monoclonal antibodies labelled only mesoderm-derived tissue components, but did not recognize vimentin in astrocytes and Bergmann glial fibers. Pre-heating formaldehyde-fixed sections from cattle, rabbit and rat brain in a microwave oven prior to the immunohistochemical reaction resulted in an enormous enhancement of vimentin staining of mesoderm-derived tissues, of astrocytes and bergmann cell fibers.

Co-expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study

Changes in the distribution of proliferating astrocytes expressing glial fibrillary acidic protein (GFAP) and/or vimentin were examined in the injured cerebral hemisphere in adult mice. The injury was followed by [3H]thymidine injections at different time intervals. The brain sections were doubly immunostained for GFAP and vimentin and subjected to autoradiography. In that way three cell types were distinguished immunocytochemically: 1.(1) astrocytes co-expressing glial fibrillary acidic protein (GFAP) and vimentin2.(2) astrocytes expressing only GFAP3.(3) astrocyte-like cells expressing vimentin. Thereafter, numbers of immunopositive and autoradiographically labelled cells and their locations within the region of injury were recorded at each stage of the experiment. Two hours as well as 1 day after the injury proliferation of GFAP-positive astrocytes and of those co-expressing GFAP and vimentin could only be seen as statistically insignificant phenomena. On day 2 the reactive proliferation of each immunocytochemically defined cell type was already maximal, then gradually decreased and its last signs were recorded on day 8. On day 2, among all the proliferating GFAP-positive astrocytes, 67.2% were also vimentin-positive. Later, the proportion declined to 50.7% and 38.5% on days 4 and 8, respectively. The labelled astrocyte-like vimentin-positive cells were located closest to the lesion margins. In comparison, the astrocytes co-expressing GFAP and vimentin and those expressing exclusively GFAP, occupied regions progressively farther from the lesion site. At the initial stages of the response to injury, vimentin expression in cells starting their reactive proliferation did not precede the expression of GFAP. This was considered as an argument against a hypothesis that reactive astrocyte division induces a two-stage increase in the cytoskeletal protein level in which synthesis of vimentin precedes that of GFAP.

Kindling causes changes in the composition of the astrocytic cytoskeleton

Changes in the astrocytic cytoskeleton were examined in amygdala kindled rats using immunocytochemical techniques. One week following kindling, there was a dramatic increase in immunoreactivity to glial fibrillary acidic protein and vimentin in astrocytes throughout amygdala, pyriform cortex and hippocampus bilaterally. Since these changes occurred in anatomical sites involved in the propagation of kindled seizures, the observed cytoskeletal reorganization in astrocytes may signify important functional alterations in the kindled brain.

Phorbol Myristate Acetate and 8‐Bromo‐Cyclic AMP‐Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquisition of vimentin in astrocytes cultured from postnatal rat brain

Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin-GFAP transition which occurs during glial development in situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.

Transition between immature radial glia and mature astrocytes studied with a monoclonal antibody to vimentin

A monoclonal antibody to vimentin (RBA1) and a polyclonal antiserum to glial fibrillary acidic protein (GFAP) were used in double labeling experiments to examine astrocyte intermediate filaments in development and wounding. RBA1 bound to radial glia in newborn rat parietal cortex that are predominantly anti-GFAP-negative. The RBA1-positive radial fibers disappeared by postnatal day 20 with the greatest rate of disappearance occurring between day 8 and day 15. Between birth and day 20, the anti-GFAP staining increased to the adult pattern in mature shaped astrocytes. Some overlay was observed between the binding patterns of the two antibodies. Stab wounds to cortical areas were made at a developmental time when there were normally no RBA1-positive astrocytes. RBA1-positivity was present in some astrocytes but only at the edges of the wounds. The distribution patterns of RBA1-positive cells led to hypotheses concerning the possible function of vimentin in astrocytes and its regulation during development and wounding.

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.