Immunohistochemical studies on the proliferation of reactive astrocytes and the expression of cytoskeletal proteins following brain injury in rats

doi:10.1016/0165-3806(88)90045-4

Developmental Brain Research

Volume 38, Issue 2, 1 February 1988, Pages 201-210

https://doi.org/10.1016/0165-3806(88)90045-4 Get rights and content

Abstract

The appearance of reactive astrocytes following brain injury was investigated in 4-week-old rats with special reference to their proliferation and chromological changes in the cytoskeletal proteins. Two days after the injury, glial fibrillary acidic protein (GFAP)-positive cells had increased in number around the lesion and spread to the entire ipsilateral cortex by 3 days after the injury. To investigate the distribution of mitotic cells and its chronological change, immunohistochemical staining with monoclonal antibody to bromodeoxyuridine (BrdU) was performed. BrdU-positive cells began to appear around the lesion and spread to the entire ipsilateral cortex by 3 days and their distribution was the same as that of GFAP-positive cells. To investigate the association of GFAP-positive cells with cell division, double labeling experiments using [³H]thymidine autoradiography and immunohistochemical staining with antiserum to GFAP were performed. Cells doubly labeled with GFAP and [³H]thymidine were localized in the area adjacent to the lesion, in the molecular layer of the cortex and in the white matter. By contrast, none of the cells were doubly labeled in the IInd to VIth layers of the cortex. Furthermore, only astrocytes in the former areas expressed vimentin transiently from 2 to 10 days after the injury. In the rats administered vincristine, cells arrested during mitosis were found in the regions which express vimentin. From these results, it was suggested that astrocytes in the molecular layer of the cortex and the white matter adjacent to the lesion proliferated in response to the injury and expressed vimentin transiently, then acquired GFAP, and that astrocytes in the IInd to VIth layers of the cortex became reactive astrocytes without mitosis.

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OTA, to the contrary, decreases GFAP expression (Zurich et al., 2005). Simultaneously, another marker for astroglial reactivity (Takamiya et al., 1988), vimentin is increased, demonstrating that astrocytes become reactive, but in an atypical manner (Zurich et al., 2005). Brain inflammation can be either neuroprotective/neuroreparative or neurodegenerative (Monnet-Tschudi et al., 2007), and this duality is reflected in the two microglial activation phenotypes (Boche et al., 2013; Schwartz et al., 2006); the alternative M2 phenotype that favours tissue repair, and the classical M1 phenotype with neurodegeneration as outcome (Kigerl et al., 2009).
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Western blot data and their semiquantitative analysis reported in panel B, and C, confirmed the morphological data showing in controls a progressive growing of CSPG expression in the 12–48-h interval followed by a decrease within the period of 72-120 h and a much reduced expression of CSPG in treated animals. Reactive astrogliosis is characterized by astrocyte morphological changes, such as cell hypertrophy and over-expression of GFAP (Eng and Ghirnikar, 1994; Takamiya et al., 1998), nevertheless the mechanism underlying this process is not yet fully identified. Our previous data showing involvement of NO, Ca2+, CaM and ERK in the signaling cascade mediating the mitogenic effect of IL-1β in human astrocytoma U-373 MG cells (Meini et al., 2006, 2008), prompted us to investigate, in the same cell line, a likely role of these agents as well as of NF-kB, a known mediator of inflammation (Aktan, 2004), in IL-1β-induced astrogliosis.
Reactive astrogliosis, a feature of neuro-inflammation is induced by a number of endogenous mediators including cytokines. Despite interleukin-1 beta (IL-1β) stands out as the major inducer of this process, the underlying mechanism and its role on neuronal viability remain elusive. We investigated in human astrocytoma cells and the rat brain striatum, the role of the nuclear factor-kB (NF-kB) intracellular Ca²⁺ concentration ([Ca²⁺]_i) calmodulin (CaM) and extracellular regulated mitogen-activated protein kinases (ERK1/2) in IL-1β-induced expression of glial fibrillary acidic protein (GFAP) and neuronal apoptosis associated to a brain trauma. Cell data showed that IL-1β (1 ng/ml) increased NF-kB, pERK1/2 and GFAP expression. Nevertheless, further increase in IL-1β levels reversed progressively these responses. Preventing ERK1/2 activation with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]-butadiene antagonized IL-1β-induced GFAP expression while inhibiting selectively nuclear translocation of NF-kB with caffeic-acid phenethyl-ester down-regulated both ERK1/2 and GFAP expression induced by IL-1β. The GFAP response was also prevented by antagonizing selectively increase in [Ca²⁺]_i, CaM activity or inducible nitric oxide synthase expression with respectively ryanodine plus 2-aminoethoxydiphenyl-borate, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride and N-[(3-(aminomethyl)-phenyl]methyl]-ethanimidamide dihydrochloride. Data in vivo supported these findings and showed that GFAP expression induced by IL-1β (50 ng/ml) correlated with attenuated glial scar formation and reduced neuronal apoptosis. Our data identified the NF-kB/Ca²⁺–CaM/ERK signaling pathway as a novel in vivo key regulator of IL-1β-induced astrogliosis which may represent a potential target in neurodegeneration.
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Research reportImmunohistochemical studies on the proliferation of reactive astrocytes and the expression of cytoskeletal proteins following brain injury in rats

Abstract

Fine structure of reactive cells in injured nervous tissue labeled with 3H-thymidine injected before injury

J. Comp. Neurol.

The proliferation of astrocytes around a needle wound in the rat brain

J. Anat.

Regulation of glial fibrillary acidic protein (GFAP) expression in CNS development and in pathological states

J. Neuroimmunol.

The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast-type filament protein, vimentin

J. Neurochem.

Origin, morphology and function of the microglia

Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine. A new reagent for detection of DNA replication

Science

Action of the vinca alkaloids vincristine, vinblastine, and desacetyl vinblastine amide on microtubules in vitro

Cancer Res.

The use of anti-avidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase techniques

Am. J. Clin. Pathol.

Dynamic aspects of glial reactions in altered brains

Pathol. Res. Pract.

Cytologic and cytochemical studies of neuroglia. III. The DNA content of giant fibrous astrocytes, with implications concerning the nature of these cells

J. Neuropathol. Exp. Neurol.