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Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure.

Madeira MH, Elvas F, Boia R, Gonçalves FQ, Cunha RA, Ambrósio AF, Santiago AR - J Neuroinflammation (2015)

Bottom Line: We report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP.Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.This work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. mariah.madeira@gmail.com.

ABSTRACT

Background: Elevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS).

Methods: Retinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies.

Results: We report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.

Conclusions: This work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

No MeSH data available.


Related in: MedlinePlus

LPS or EHP increases A2AR expression and density in retinal microglia and increase the extracellular ATP levels. Retinal organotypic cultures were challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 24 h. a A2AR mRNA expression was assayed by qPCR. Results are presented as fold change of the control, from six to ten independent experiments. b Organotypic retinal cultures were immunostained for A2AR (grey/green; arrowheads) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). Representative images obtained from four independent experiments. c The extracellular levels of ATP in the medium were quantified by luciferin-luciferase ATP-dependent reaction. Results are expressed as percentage of control and are mean ± SEM of six to eight independent experiments. **P < 0.01, different from control; Kruskal-Wallis test, followed by Dunn’s multiple comparison test
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Fig1: LPS or EHP increases A2AR expression and density in retinal microglia and increase the extracellular ATP levels. Retinal organotypic cultures were challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 24 h. a A2AR mRNA expression was assayed by qPCR. Results are presented as fold change of the control, from six to ten independent experiments. b Organotypic retinal cultures were immunostained for A2AR (grey/green; arrowheads) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). Representative images obtained from four independent experiments. c The extracellular levels of ATP in the medium were quantified by luciferin-luciferase ATP-dependent reaction. Results are expressed as percentage of control and are mean ± SEM of six to eight independent experiments. **P < 0.01, different from control; Kruskal-Wallis test, followed by Dunn’s multiple comparison test

Mentions: LPS or EHP exposure for 4 h significantly increased A2AR messenger RNA (mRNA) expression in the retina by 5.3- and 6.0-fold (n = 6–10), respectively (Fig. 1a). Accordingly, 4 h after exposure to LPS or EHP, A2AR immunoreactivity increased mainly in CD11b-positive cells in the GCL (Fig. 1b), indicating that A2AR in the GCL are mainly present in microglia.Fig. 1


Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure.

Madeira MH, Elvas F, Boia R, Gonçalves FQ, Cunha RA, Ambrósio AF, Santiago AR - J Neuroinflammation (2015)

LPS or EHP increases A2AR expression and density in retinal microglia and increase the extracellular ATP levels. Retinal organotypic cultures were challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 24 h. a A2AR mRNA expression was assayed by qPCR. Results are presented as fold change of the control, from six to ten independent experiments. b Organotypic retinal cultures were immunostained for A2AR (grey/green; arrowheads) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). Representative images obtained from four independent experiments. c The extracellular levels of ATP in the medium were quantified by luciferin-luciferase ATP-dependent reaction. Results are expressed as percentage of control and are mean ± SEM of six to eight independent experiments. **P < 0.01, different from control; Kruskal-Wallis test, followed by Dunn’s multiple comparison test
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465153&req=5

Fig1: LPS or EHP increases A2AR expression and density in retinal microglia and increase the extracellular ATP levels. Retinal organotypic cultures were challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 24 h. a A2AR mRNA expression was assayed by qPCR. Results are presented as fold change of the control, from six to ten independent experiments. b Organotypic retinal cultures were immunostained for A2AR (grey/green; arrowheads) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). Representative images obtained from four independent experiments. c The extracellular levels of ATP in the medium were quantified by luciferin-luciferase ATP-dependent reaction. Results are expressed as percentage of control and are mean ± SEM of six to eight independent experiments. **P < 0.01, different from control; Kruskal-Wallis test, followed by Dunn’s multiple comparison test
Mentions: LPS or EHP exposure for 4 h significantly increased A2AR messenger RNA (mRNA) expression in the retina by 5.3- and 6.0-fold (n = 6–10), respectively (Fig. 1a). Accordingly, 4 h after exposure to LPS or EHP, A2AR immunoreactivity increased mainly in CD11b-positive cells in the GCL (Fig. 1b), indicating that A2AR in the GCL are mainly present in microglia.Fig. 1

Bottom Line: We report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP.Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.This work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. mariah.madeira@gmail.com.

ABSTRACT

Background: Elevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS).

Methods: Retinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies.

Results: We report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.

Conclusions: This work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

No MeSH data available.


Related in: MedlinePlus