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Microglia activation is essential for BMP7-mediated retinal reactive gliosis

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.


Related in: MedlinePlus

PLX ablates microglia in the retina. Mice were fed with chow-containing PLX or vehicle dye to determine ablation of microglia in the retina. a Schematic describing the time points for which the mice were fed with chow-containing PLX, following which eyes were harvested. Retinal flatmounts prepared from the eyes harvested at 7, 14, and 21 days were labeled for GFAP or IBA1 (b–i). Insets in b–e indicate the flatmount outline and from where the images (b–i) was taken. While GFAP did not show any difference between the stages examine (b–e), there was a significant decrease in IBA1 label in mice kept on PLX diet for 7 days (f, g). By 14 days, no IBA1 label was found in the retinal flatmount and this absence persisted into the 21-day time point (h, i). Retinal sections control and PLX-treated mice labeled for IBA1 to show loss of microglia in the deeper layers of the retina (j–m). Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Magnification bar in b = 50 μm, for images b–i. Magnification bar in j = 50 μm, for images j–m. Abbreviations: GFAP glial fibrillary acidic protein, IBA1 ionized calcium-binding adapter molecule 1
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Fig5: PLX ablates microglia in the retina. Mice were fed with chow-containing PLX or vehicle dye to determine ablation of microglia in the retina. a Schematic describing the time points for which the mice were fed with chow-containing PLX, following which eyes were harvested. Retinal flatmounts prepared from the eyes harvested at 7, 14, and 21 days were labeled for GFAP or IBA1 (b–i). Insets in b–e indicate the flatmount outline and from where the images (b–i) was taken. While GFAP did not show any difference between the stages examine (b–e), there was a significant decrease in IBA1 label in mice kept on PLX diet for 7 days (f, g). By 14 days, no IBA1 label was found in the retinal flatmount and this absence persisted into the 21-day time point (h, i). Retinal sections control and PLX-treated mice labeled for IBA1 to show loss of microglia in the deeper layers of the retina (j–m). Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Magnification bar in b = 50 μm, for images b–i. Magnification bar in j = 50 μm, for images j–m. Abbreviations: GFAP glial fibrillary acidic protein, IBA1 ionized calcium-binding adapter molecule 1

Mentions: To further investigate the role of microglia in BMP7-mediated gliosis, a means to ablate microglial cells within the retina was sought. Previous reports have shown colony stimulating factor receptor 1 (CSFR1) inhibitor, PLX3397, to selectively ablate microglia in the brain [49]. We have used a variant of the drug, PLX5622, supplied by Plexxikon Inc. in chow form to determine its effect on retinal microglia. Starting at postnatal day 30, mice were switched to control chow or chow containing 1200 ppm PLX. The mice continued treatment with the inhibitor-laced chow until sacrificed 7, 14, or 21 days later. Retinal flatmounts from control and PLX mice were isolated for 7, 14, and 21 days, and labeled for IBA1 and GFAP (Fig. 5). Although no apparent change in GFAP was observed (Fig. 5b–e), there was a clear decrease in the number of IBA1+ cells 7 days after starting the PLX diet, and IBA1 immunoreactivity was completely lost by 14 days (Fig. 5f–m). Retinal tissue sections from these mice were also analyzed for ganglion cells (Brn3a), bipolar cells (Chx10), Müller glia (SOX9), and horizontal cells (Calbindin) (Fig. 6a–d, f–i). Cell counts for labeled cells showed no statistically significant change between the control and PLX-treated mice (Fig. 6k). Retinal flatmounts of PLX and vehicle-treated retinas were also labeled with Gα transducin to label photoreceptors (Fig. 6e, j). Cell count of labeled images showed no statistically significant change in cell numbers (Fig. 6l). Thickness of retinal sections of the control and PLX treated mice also showed no change (Fig. 6m).Fig. 5


Microglia activation is essential for BMP7-mediated retinal reactive gliosis
PLX ablates microglia in the retina. Mice were fed with chow-containing PLX or vehicle dye to determine ablation of microglia in the retina. a Schematic describing the time points for which the mice were fed with chow-containing PLX, following which eyes were harvested. Retinal flatmounts prepared from the eyes harvested at 7, 14, and 21 days were labeled for GFAP or IBA1 (b–i). Insets in b–e indicate the flatmount outline and from where the images (b–i) was taken. While GFAP did not show any difference between the stages examine (b–e), there was a significant decrease in IBA1 label in mice kept on PLX diet for 7 days (f, g). By 14 days, no IBA1 label was found in the retinal flatmount and this absence persisted into the 21-day time point (h, i). Retinal sections control and PLX-treated mice labeled for IBA1 to show loss of microglia in the deeper layers of the retina (j–m). Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Magnification bar in b = 50 μm, for images b–i. Magnification bar in j = 50 μm, for images j–m. Abbreviations: GFAP glial fibrillary acidic protein, IBA1 ionized calcium-binding adapter molecule 1
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Fig5: PLX ablates microglia in the retina. Mice were fed with chow-containing PLX or vehicle dye to determine ablation of microglia in the retina. a Schematic describing the time points for which the mice were fed with chow-containing PLX, following which eyes were harvested. Retinal flatmounts prepared from the eyes harvested at 7, 14, and 21 days were labeled for GFAP or IBA1 (b–i). Insets in b–e indicate the flatmount outline and from where the images (b–i) was taken. While GFAP did not show any difference between the stages examine (b–e), there was a significant decrease in IBA1 label in mice kept on PLX diet for 7 days (f, g). By 14 days, no IBA1 label was found in the retinal flatmount and this absence persisted into the 21-day time point (h, i). Retinal sections control and PLX-treated mice labeled for IBA1 to show loss of microglia in the deeper layers of the retina (j–m). Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Magnification bar in b = 50 μm, for images b–i. Magnification bar in j = 50 μm, for images j–m. Abbreviations: GFAP glial fibrillary acidic protein, IBA1 ionized calcium-binding adapter molecule 1
Mentions: To further investigate the role of microglia in BMP7-mediated gliosis, a means to ablate microglial cells within the retina was sought. Previous reports have shown colony stimulating factor receptor 1 (CSFR1) inhibitor, PLX3397, to selectively ablate microglia in the brain [49]. We have used a variant of the drug, PLX5622, supplied by Plexxikon Inc. in chow form to determine its effect on retinal microglia. Starting at postnatal day 30, mice were switched to control chow or chow containing 1200 ppm PLX. The mice continued treatment with the inhibitor-laced chow until sacrificed 7, 14, or 21 days later. Retinal flatmounts from control and PLX mice were isolated for 7, 14, and 21 days, and labeled for IBA1 and GFAP (Fig. 5). Although no apparent change in GFAP was observed (Fig. 5b–e), there was a clear decrease in the number of IBA1+ cells 7 days after starting the PLX diet, and IBA1 immunoreactivity was completely lost by 14 days (Fig. 5f–m). Retinal tissue sections from these mice were also analyzed for ganglion cells (Brn3a), bipolar cells (Chx10), Müller glia (SOX9), and horizontal cells (Calbindin) (Fig. 6a–d, f–i). Cell counts for labeled cells showed no statistically significant change between the control and PLX-treated mice (Fig. 6k). Retinal flatmounts of PLX and vehicle-treated retinas were also labeled with Gα transducin to label photoreceptors (Fig. 6e, j). Cell count of labeled images showed no statistically significant change in cell numbers (Fig. 6l). Thickness of retinal sections of the control and PLX treated mice also showed no change (Fig. 6m).Fig. 5

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 M&uuml;ller 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&nbsp;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.


Related in: MedlinePlus