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Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants.

Ferrer-Martín RM, Martín-Oliva D, Sierra-Martín A, Carrasco MC, Martín-Estebané M, Calvente R, Martín-Guerrero SM, Marín-Teva JL, Navascués J, Cuadros MA - PLoS ONE (2015)

Bottom Line: Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles.The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution.Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.

ABSTRACT
The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.

No MeSH data available.


Related in: MedlinePlus

Microglial distribution in minocycline-treated and untreated explants.The left panel shows microglial cells (stained with anti-CD45 antibody, green), whereas the right panel depicts both microglial cells and the location of retinal layers (revealed by nuclear staining with Hoechst, blue). A. Microglial cells in the control explants (CT) are frequently seen within the Outer Nuclear Layer (ONL). B. In minocycline-treated explants (MIN), microglial cells are more compact and do not penetrate the ONL. Representative images of at least three different explants per condition. INL, Inner Nuclear Layer; GCL, Ganglion Cell Layer. Scale bar, 50 μm.
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pone.0135238.g002: Microglial distribution in minocycline-treated and untreated explants.The left panel shows microglial cells (stained with anti-CD45 antibody, green), whereas the right panel depicts both microglial cells and the location of retinal layers (revealed by nuclear staining with Hoechst, blue). A. Microglial cells in the control explants (CT) are frequently seen within the Outer Nuclear Layer (ONL). B. In minocycline-treated explants (MIN), microglial cells are more compact and do not penetrate the ONL. Representative images of at least three different explants per condition. INL, Inner Nuclear Layer; GCL, Ganglion Cell Layer. Scale bar, 50 μm.

Mentions: Minocycline treatment also modified the morphological features, distribution, and activation state of microglial cells. These had a honeycomb-like swollen appearance in untreated explants (Fig 2A) and acquired a more compact morphology in minocycline-treated explants (Fig 2B), suggesting a lower state of activation. Moreover, microglial cells entered the ONL in control explants but not in minocycline-treated explants (Fig 2A and 2B). Given that most cell death was detected at the ONL (see Fig 1C) and that a portion of dead/dying cells is reported to be engulfed by microglial cells in the ONL [31], minocycline treatment resulted in abundant non-phagocytosed debris (S3 Fig).


Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants.

Ferrer-Martín RM, Martín-Oliva D, Sierra-Martín A, Carrasco MC, Martín-Estebané M, Calvente R, Martín-Guerrero SM, Marín-Teva JL, Navascués J, Cuadros MA - PLoS ONE (2015)

Microglial distribution in minocycline-treated and untreated explants.The left panel shows microglial cells (stained with anti-CD45 antibody, green), whereas the right panel depicts both microglial cells and the location of retinal layers (revealed by nuclear staining with Hoechst, blue). A. Microglial cells in the control explants (CT) are frequently seen within the Outer Nuclear Layer (ONL). B. In minocycline-treated explants (MIN), microglial cells are more compact and do not penetrate the ONL. Representative images of at least three different explants per condition. INL, Inner Nuclear Layer; GCL, Ganglion Cell Layer. Scale bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4529135&req=5

pone.0135238.g002: Microglial distribution in minocycline-treated and untreated explants.The left panel shows microglial cells (stained with anti-CD45 antibody, green), whereas the right panel depicts both microglial cells and the location of retinal layers (revealed by nuclear staining with Hoechst, blue). A. Microglial cells in the control explants (CT) are frequently seen within the Outer Nuclear Layer (ONL). B. In minocycline-treated explants (MIN), microglial cells are more compact and do not penetrate the ONL. Representative images of at least three different explants per condition. INL, Inner Nuclear Layer; GCL, Ganglion Cell Layer. Scale bar, 50 μm.
Mentions: Minocycline treatment also modified the morphological features, distribution, and activation state of microglial cells. These had a honeycomb-like swollen appearance in untreated explants (Fig 2A) and acquired a more compact morphology in minocycline-treated explants (Fig 2B), suggesting a lower state of activation. Moreover, microglial cells entered the ONL in control explants but not in minocycline-treated explants (Fig 2A and 2B). Given that most cell death was detected at the ONL (see Fig 1C) and that a portion of dead/dying cells is reported to be engulfed by microglial cells in the ONL [31], minocycline treatment resulted in abundant non-phagocytosed debris (S3 Fig).

Bottom Line: Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles.The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution.Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.

ABSTRACT
The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.

No MeSH data available.


Related in: MedlinePlus