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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

Blockade of A2AR decreases the expression and immunoreactivity of iNOS and NO production induced by LPS or EHP. Retinal organotypic cultures were pretreated with SCH 58261 (50 nM) and then challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 4 h. a iNOS mRNA expression was assessed by qPCR. Results are presented as fold change of the control, from six to twelve independent experiments. b Organotypic retinal cultures were immunostained for iNOS (green) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). The images are representative of four to five independent experiments. c The immunoreactivity of iNOS in microglia localized in the GCL was quantified. Results are expressed as percentage of control from four to five independent experiments. d The production of NO was assessed by the Griess reaction in the culture supernatants, and nitrite formation was quantified. Results are expressed as percentage of control and are mean ± SEM of four to six independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, different from control; +P < 0.05, different from LPS; #P < 0.05 and ###P < 0.001, different from EHP; Kruskal-Wallis test, followed by Dunn’s multiple comparison test
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Fig3: Blockade of A2AR decreases the expression and immunoreactivity of iNOS and NO production induced by LPS or EHP. Retinal organotypic cultures were pretreated with SCH 58261 (50 nM) and then challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 4 h. a iNOS mRNA expression was assessed by qPCR. Results are presented as fold change of the control, from six to twelve independent experiments. b Organotypic retinal cultures were immunostained for iNOS (green) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). The images are representative of four to five independent experiments. c The immunoreactivity of iNOS in microglia localized in the GCL was quantified. Results are expressed as percentage of control from four to five independent experiments. d The production of NO was assessed by the Griess reaction in the culture supernatants, and nitrite formation was quantified. Results are expressed as percentage of control and are mean ± SEM of four to six independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, different from control; +P < 0.05, different from LPS; #P < 0.05 and ###P < 0.001, different from EHP; Kruskal-Wallis test, followed by Dunn’s multiple comparison test

Mentions: As expected, the mRNA expression of iNOS significantly increased by 30.5-fold after 4 h of exposure to LPS (n = 6), and this effect was significantly decreased upon A2AR blockade (n = 4) (Fig. 3a). The exposure to EHP for 4 h also significantly increased iNOS mRNA expression by 4.6-fold over control (n = 5), and the blockade of A2AR also significantly prevented this effect (n = 6) (Fig. 3a).Fig. 3


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)

Blockade of A2AR decreases the expression and immunoreactivity of iNOS and NO production induced by LPS or EHP. Retinal organotypic cultures were pretreated with SCH 58261 (50 nM) and then challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 4 h. a iNOS mRNA expression was assessed by qPCR. Results are presented as fold change of the control, from six to twelve independent experiments. b Organotypic retinal cultures were immunostained for iNOS (green) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). The images are representative of four to five independent experiments. c The immunoreactivity of iNOS in microglia localized in the GCL was quantified. Results are expressed as percentage of control from four to five independent experiments. d The production of NO was assessed by the Griess reaction in the culture supernatants, and nitrite formation was quantified. Results are expressed as percentage of control and are mean ± SEM of four to six independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, different from control; +P < 0.05, different from LPS; #P < 0.05 and ###P < 0.001, different from EHP; Kruskal-Wallis test, followed by Dunn’s multiple comparison test
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Blockade of A2AR decreases the expression and immunoreactivity of iNOS and NO production induced by LPS or EHP. Retinal organotypic cultures were pretreated with SCH 58261 (50 nM) and then challenged with LPS (3 μg/mL) or EHP (+70 mmHg) for 4 h. a iNOS mRNA expression was assessed by qPCR. Results are presented as fold change of the control, from six to twelve independent experiments. b Organotypic retinal cultures were immunostained for iNOS (green) and CD11b (microglia marker; red) and imaged in the GCL using a confocal microscope. Nuclei were stained with DAPI (blue). The images are representative of four to five independent experiments. c The immunoreactivity of iNOS in microglia localized in the GCL was quantified. Results are expressed as percentage of control from four to five independent experiments. d The production of NO was assessed by the Griess reaction in the culture supernatants, and nitrite formation was quantified. Results are expressed as percentage of control and are mean ± SEM of four to six independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, different from control; +P < 0.05, different from LPS; #P < 0.05 and ###P < 0.001, different from EHP; Kruskal-Wallis test, followed by Dunn’s multiple comparison test
Mentions: As expected, the mRNA expression of iNOS significantly increased by 30.5-fold after 4 h of exposure to LPS (n = 6), and this effect was significantly decreased upon A2AR blockade (n = 4) (Fig. 3a). The exposure to EHP for 4 h also significantly increased iNOS mRNA expression by 4.6-fold over control (n = 5), and the blockade of A2AR also significantly prevented this effect (n = 6) (Fig. 3a).Fig. 3

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