Dataset Information

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo.

ABSTRACT:

Background

Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin(+) cells. Thus, the present study evaluates expression of nestin and other neural markers in quiescent and proliferating microglia in naïve retina and following ON transection in adult rats in vivo.

Methodology/principal findings

For analysis of cell proliferation and cells fates, rats received BrdU injections. Microglia in retinal sections or isolated cells were characterized using immunofluorescence labeling with markers for microglia (e.g., Iba1, CD11b), cell proliferation, and neural cells (e.g., nestin, vimentin, NG2, GFAP, Doublecortin etc.). Cellular analyses were performed using confocal laser scanning microscopy. In the naïve adult rat retina, about 60% of resting ramified microglia expressed nestin. After ON transection, numbers of nestin(+) microglia peaked to a maximum at 7 days, primarily due to in situ cell proliferation of exclusively nestin(+) microglia. After 8 weeks, microglia numbers re-attained control levels, but 20% were still BrdU(+) and nestin(+), although no further local cell proliferation occurred. In addition, nestin(+) microglia co-expressed vimentin and NG2, but not GFAP or neuronal markers. Fourteen days after injury and following retrograde labeling of retinal ganglion cells (RGCs) with Fluorogold (FG), nestin(+)NG2(+) microglia were positive for the dye indicating an active involvement of a proliferating cell population in phagocytosing apoptotic retinal neurons.

Conclusions/significance

The current study provides evidence that in adult rat retina, a specific resident population of microglia expresses proteins of immature neural cells that are involved in injury-induced cell proliferation and phagocytosis while transdifferentiation was not observed.

SUBMITTER: Wohl SG

PROVIDER: S-EPMC3151247 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Publications

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo.

Wohl Stefanie G SG Schmeer Christian W CW Friese Thomas T Witte Otto W OW Isenmann Stefan S

PloS one 20110805 8

<h4>Background</h4>Following injury, microglia become activated with subsets expressing nestin as well as other neural markers. Moreover, cerebral microglia can give rise to neurons in vitro. In a previous study, we analysed the proliferation potential and nestin re-expression of retinal macroglial cells such as astrocytes and Müller cells after optic nerve (ON) lesion. However, we were unable to identify the majority of proliferative nestin(+) cells. Thus, the present study evaluates expression ...[more]

PMID: 21850226

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Project description:To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury.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.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.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.

Project description:PurposeChoroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells.MethodsWe used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF).ResultsIn the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels.ConclusionsOur results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.

Dataset Information

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo.

Background

Methodology/principal findings

Conclusions/significance

Publications

In situ dividing and phagocytosing retinal microglia express nestin, vimentin, and NG2 in vivo.

Similar Datasets

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