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Microglia activation is essential for BMP7-mediated retinal reactive gliosis

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ABSTRACT

Background: Our previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.

Methods: Adult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.

Results: Mice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.

Conclusions: Gliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0855-0) contains supplementary material, which is available to authorized users.

No MeSH data available.


BMP7 alters microglial morphology. Retinal flatmounts from 1 day BMP7- and vehicle-injected retina were labeled for IBA1 (a, b) and analyzed for morphological changes. An increase in the area of the microglia was observed when compared to the vehicle control (c). Number of branches and branch length were also assessed for the treated cells, and increase in the number of branches was observed with a decrease in the branch length of the cells incubated with LPS or BMP7 when compared to the vehicle control (c). Data shown in c represent relative change in area and number of branches in BMP7-treated samples to the vehicle control. Bars above a level of 1.0 (solid black line) represent an increase while bars below the level of 1.0 represent a decrease in the parameter measured, relative to the corresponding vehicle control. Magnification bar in a = 50 μm, for images (a) and (b). Abbreviation: IBA1 ionized calcium-binding adapter molecule 1
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Fig3: BMP7 alters microglial morphology. Retinal flatmounts from 1 day BMP7- and vehicle-injected retina were labeled for IBA1 (a, b) and analyzed for morphological changes. An increase in the area of the microglia was observed when compared to the vehicle control (c). Number of branches and branch length were also assessed for the treated cells, and increase in the number of branches was observed with a decrease in the branch length of the cells incubated with LPS or BMP7 when compared to the vehicle control (c). Data shown in c represent relative change in area and number of branches in BMP7-treated samples to the vehicle control. Bars above a level of 1.0 (solid black line) represent an increase while bars below the level of 1.0 represent a decrease in the parameter measured, relative to the corresponding vehicle control. Magnification bar in a = 50 μm, for images (a) and (b). Abbreviation: IBA1 ionized calcium-binding adapter molecule 1

Mentions: Changes in morphological characteristics of microglia following control and BMP7 treatments were subsequently investigated. It has been reported by other investigators that activated microglia increase in area with an increase in branch points [48]. Retinal flatmounts of 1 day BMP7- and vehicle-treated retinas were labeled with IBA1 and analyzed for average cell area and number of branch points in cellular processes (Fig. 3a, b). Graphs show relative changes in the area and number of branches (“Median intersections” output from the Sholl analysis). Morphological analysis revealed that the BMP7-treated retinas contained microglia with a larger area in comparison to vehicle-treated retinas, and a decrease in the number of branches (Fig. 3c).Fig. 3


Microglia activation is essential for BMP7-mediated retinal reactive gliosis
BMP7 alters microglial morphology. Retinal flatmounts from 1 day BMP7- and vehicle-injected retina were labeled for IBA1 (a, b) and analyzed for morphological changes. An increase in the area of the microglia was observed when compared to the vehicle control (c). Number of branches and branch length were also assessed for the treated cells, and increase in the number of branches was observed with a decrease in the branch length of the cells incubated with LPS or BMP7 when compared to the vehicle control (c). Data shown in c represent relative change in area and number of branches in BMP7-treated samples to the vehicle control. Bars above a level of 1.0 (solid black line) represent an increase while bars below the level of 1.0 represent a decrease in the parameter measured, relative to the corresponding vehicle control. Magnification bar in a = 50 μm, for images (a) and (b). Abbreviation: IBA1 ionized calcium-binding adapter molecule 1
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Fig3: BMP7 alters microglial morphology. Retinal flatmounts from 1 day BMP7- and vehicle-injected retina were labeled for IBA1 (a, b) and analyzed for morphological changes. An increase in the area of the microglia was observed when compared to the vehicle control (c). Number of branches and branch length were also assessed for the treated cells, and increase in the number of branches was observed with a decrease in the branch length of the cells incubated with LPS or BMP7 when compared to the vehicle control (c). Data shown in c represent relative change in area and number of branches in BMP7-treated samples to the vehicle control. Bars above a level of 1.0 (solid black line) represent an increase while bars below the level of 1.0 represent a decrease in the parameter measured, relative to the corresponding vehicle control. Magnification bar in a = 50 μm, for images (a) and (b). Abbreviation: IBA1 ionized calcium-binding adapter molecule 1
Mentions: Changes in morphological characteristics of microglia following control and BMP7 treatments were subsequently investigated. It has been reported by other investigators that activated microglia increase in area with an increase in branch points [48]. Retinal flatmounts of 1 day BMP7- and vehicle-treated retinas were labeled with IBA1 and analyzed for average cell area and number of branch points in cellular processes (Fig. 3a, b). Graphs show relative changes in the area and number of branches (“Median intersections” output from the Sholl analysis). Morphological analysis revealed that the BMP7-treated retinas contained microglia with a larger area in comparison to vehicle-treated retinas, and a decrease in the number of branches (Fig. 3c).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Our previous studies have shown that BMP7 is able to trigger activation of retinal macroglia. However, these studies showed the responsiveness of Müller glial cells and retinal astrocytes in vitro was attenuated in comparison to those in vivo, indicating other retinal cell types may be mediating the response of the macroglial cells to BMP7. In this study, we test the hypothesis that BMP7-mediated gliosis is the result of inflammatory signaling from retinal microglia.

Methods: Adult mice were injected intravitreally with BMP7 and eyes harvested 1, 3, or 7 days postinjection. Some mice were treated with PLX5622 (PLX) to ablate microglia and were subsequently injected with control or BMP7. Processed tissue was analyzed via immunofluorescence, RT-qPCR, or ELISA. In addition, cultures of retinal microglia were treated with vehicle, lipopolysaccharide, or BMP7 to determine the effects of BMP7-isolated cells.

Results: Mice injected with BMP7 showed regulation of various inflammatory markers at the RNA level, as well as changes in microglial morphology. Isolated retinal microglia also showed an upregulation of BMP-signaling components following treatment. In vitro treatment of retinal astrocytes with conditioned media from activated microglia upregulated RNA levels of gliosis markers. In the absence of microglia, the mouse retina showed a subdued gliosis and inflammatory response when exposed to BMP7.

Conclusions: Gliosis resulting from BMP7 is mediated through an inflammatory response from retinal microglia.

Electronic supplementary material: The online version of this article (doi:10.1186/s12974-017-0855-0) contains supplementary material, which is available to authorized users.

No MeSH data available.