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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

LPS treatment does not modify the distribution of microglial cell and cell viability.A, B: Distribution of microglial cells in control (A, CT) and LPS-treated (B, LPS) retinal explants. Left panels display microglial cells labeled with anti-CD45 antibody, while right panels also show cell nuclei stained with Hoechst. The distribution of microglia in LPS-treated explants is similar to that of untreated explants. Representative images of 3 different explants per condition. Scale bar, 50 μm. C: Bar graph showing that LPS treatment has no significant effect on cell viability in retinal explants. Bars represent mean values ± SEM of 5 explants).
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pone.0135238.g006: LPS treatment does not modify the distribution of microglial cell and cell viability.A, B: Distribution of microglial cells in control (A, CT) and LPS-treated (B, LPS) retinal explants. Left panels display microglial cells labeled with anti-CD45 antibody, while right panels also show cell nuclei stained with Hoechst. The distribution of microglia in LPS-treated explants is similar to that of untreated explants. Representative images of 3 different explants per condition. Scale bar, 50 μm. C: Bar graph showing that LPS treatment has no significant effect on cell viability in retinal explants. Bars represent mean values ± SEM of 5 explants).

Mentions: Incubation of retinal explants in the presence of LPS induced a greater release of TNF-α in the culture media (Fig 5A) and augmented the proportion of proliferating (Ki67-positive) microglia (Fig 5B). These results support the view that treatment with LPS increases the activation state of microglial cells in the retinal explants. By contrast, immunocytochemical staining with anti-PAR antibody and the activated appearance of microglial cells were similar to that seen in control explants (Fig 5C and 5D; compare with Fig 3). In addition, LPS treatment had no appreciable effect on the distribution of microglial cells, which were located in all retinal layers, including the ONL, in both LPS-treated and non-treated retinal explants (Fig 6A and 6B). Therefore, the treatment with LPS apparently heightened the activation state of microglial cells which was already present, although at a lower level, in non-treated retinal explants. This higher activation level of microglial cells did not affect the cell viability in the LPS-treated explants (Fig 6C).


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)

LPS treatment does not modify the distribution of microglial cell and cell viability.A, B: Distribution of microglial cells in control (A, CT) and LPS-treated (B, LPS) retinal explants. Left panels display microglial cells labeled with anti-CD45 antibody, while right panels also show cell nuclei stained with Hoechst. The distribution of microglia in LPS-treated explants is similar to that of untreated explants. Representative images of 3 different explants per condition. Scale bar, 50 μm. C: Bar graph showing that LPS treatment has no significant effect on cell viability in retinal explants. Bars represent mean values ± SEM of 5 explants).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4529135&req=5

pone.0135238.g006: LPS treatment does not modify the distribution of microglial cell and cell viability.A, B: Distribution of microglial cells in control (A, CT) and LPS-treated (B, LPS) retinal explants. Left panels display microglial cells labeled with anti-CD45 antibody, while right panels also show cell nuclei stained with Hoechst. The distribution of microglia in LPS-treated explants is similar to that of untreated explants. Representative images of 3 different explants per condition. Scale bar, 50 μm. C: Bar graph showing that LPS treatment has no significant effect on cell viability in retinal explants. Bars represent mean values ± SEM of 5 explants).
Mentions: Incubation of retinal explants in the presence of LPS induced a greater release of TNF-α in the culture media (Fig 5A) and augmented the proportion of proliferating (Ki67-positive) microglia (Fig 5B). These results support the view that treatment with LPS increases the activation state of microglial cells in the retinal explants. By contrast, immunocytochemical staining with anti-PAR antibody and the activated appearance of microglial cells were similar to that seen in control explants (Fig 5C and 5D; compare with Fig 3). In addition, LPS treatment had no appreciable effect on the distribution of microglial cells, which were located in all retinal layers, including the ONL, in both LPS-treated and non-treated retinal explants (Fig 6A and 6B). Therefore, the treatment with LPS apparently heightened the activation state of microglial cells which was already present, although at a lower level, in non-treated retinal explants. This higher activation level of microglial cells did not affect the cell viability in the LPS-treated explants (Fig 6C).

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