Cargando…

Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin

PURPOSE: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. METHODS: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days’ duration. Injuries were induced in the right eyes of Long E...

Descripción completa

Detalles Bibliográficos
Autores principales: Luna, Gabriel, Lewis, Geoffrey P., Banna, Christopher D., Skalli, Omar, Fisher, Steven K.
Formato: Texto
Lenguaje:English
Publicado: Molecular Vision 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2997333/
https://www.ncbi.nlm.nih.gov/pubmed/21139996
_version_ 1782193294490468352
author Luna, Gabriel
Lewis, Geoffrey P.
Banna, Christopher D.
Skalli, Omar
Fisher, Steven K.
author_facet Luna, Gabriel
Lewis, Geoffrey P.
Banna, Christopher D.
Skalli, Omar
Fisher, Steven K.
author_sort Luna, Gabriel
collection PubMed
description PURPOSE: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. METHODS: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days’ duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti–glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT–PCR) techniques. RESULTS: Immunocytochemistry showed increased Müller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Müller cells. Nestin was consistently observed in the foremost processes of those Müller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Müller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Müller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. CONCLUSIONS: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Müller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Müller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries.
format Text
id pubmed-2997333
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher Molecular Vision
record_format MEDLINE/PubMed
spelling pubmed-29973332010-12-06 Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin Luna, Gabriel Lewis, Geoffrey P. Banna, Christopher D. Skalli, Omar Fisher, Steven K. Mol Vis Research Article PURPOSE: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. METHODS: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days’ duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti–glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT–PCR) techniques. RESULTS: Immunocytochemistry showed increased Müller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Müller cells. Nestin was consistently observed in the foremost processes of those Müller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Müller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Müller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. CONCLUSIONS: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Müller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Müller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries. Molecular Vision 2010-11-27 /pmc/articles/PMC2997333/ /pubmed/21139996 Text en Copyright © 2010 Molecular Vision. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Luna, Gabriel
Lewis, Geoffrey P.
Banna, Christopher D.
Skalli, Omar
Fisher, Steven K.
Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
title Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
title_full Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
title_fullStr Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
title_full_unstemmed Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
title_short Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
title_sort expression profiles of nestin and synemin in reactive astrocytes and müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2997333/
https://www.ncbi.nlm.nih.gov/pubmed/21139996
work_keys_str_mv AT lunagabriel expressionprofilesofnestinandsynemininreactiveastrocytesandmullercellsfollowingretinalinjuryacomparisonwithglialfibrillaracidicproteinandvimentin
AT lewisgeoffreyp expressionprofilesofnestinandsynemininreactiveastrocytesandmullercellsfollowingretinalinjuryacomparisonwithglialfibrillaracidicproteinandvimentin
AT bannachristopherd expressionprofilesofnestinandsynemininreactiveastrocytesandmullercellsfollowingretinalinjuryacomparisonwithglialfibrillaracidicproteinandvimentin
AT skalliomar expressionprofilesofnestinandsynemininreactiveastrocytesandmullercellsfollowingretinalinjuryacomparisonwithglialfibrillaracidicproteinandvimentin
AT fisherstevenk expressionprofilesofnestinandsynemininreactiveastrocytesandmullercellsfollowingretinalinjuryacomparisonwithglialfibrillaracidicproteinandvimentin