title: Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure.
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.
authors.affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. mariah.madeira@gmail.com.|CNC.IBILI, University of Coimbra, 3004-517, Coimbra, Portugal. mariah.madeira@gmail.com.
authors.forename: Maria H
authors.initials: MH
authors.lastname: Madeira
authors.affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. filipe.elvas@gmail.com.
authors.forename: Filipe
authors.initials: F
authors.lastname: Elvas
authors.affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. raquelfboia@gmail.com.|CNC.IBILI, University of Coimbra, 3004-517, Coimbra, Portugal. raquelfboia@gmail.com.
authors.forename: Raquel
authors.initials: R
authors.lastname: Boia
authors.affiliation: CNC.IBILI, University of Coimbra, 3004-517, Coimbra, Portugal. francisco.queiroz.goncalves@gmail.com.|CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal. francisco.queiroz.goncalves@gmail.com.
authors.forename: Francisco Q
authors.initials: FQ
authors.lastname: Gonçalves
authors.affiliation: CNC.IBILI, University of Coimbra, 3004-517, Coimbra, Portugal. cunharod@gmail.com.|CNC-Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal. cunharod@gmail.com.|Faculty of Medicine, University of Coimbra, 3000-548, Coimbra, Portugal. cunharod@gmail.com.
authors.forename: Rodrigo A
authors.initials: RA
authors.lastname: Cunha
authors.affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. afambrosio@fmed.uc.pt.|CNC.IBILI, University of Coimbra, 3004-517, Coimbra, Portugal. afambrosio@fmed.uc.pt.|Association for Innovation and Biomedical Research on Light (AIBILI), 3000-548, Coimbra, Portugal. afambrosio@fmed.uc.pt.
authors.forename: António Francisco
authors.initials: AF
authors.lastname: Ambrósio
authors.affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548, Coimbra, Portugal. asantiago@fmed.uc.pt.|CNC.IBILI, University of Coimbra, 3004-517, Coimbra, Portugal. asantiago@fmed.uc.pt.|Association for Innovation and Biomedical Research on Light (AIBILI), 3000-548, Coimbra, Portugal. asantiago@fmed.uc.pt.|Faculty of Medicine, University of Coimbra, 3000-548, Coimbra, Portugal. asantiago@fmed.uc.pt.|IBILI, Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba, 3004-548, Coimbra, Portugal. asantiago@fmed.uc.pt.
authors.forename: Ana Raquel
authors.initials: AR
authors.lastname: Santiago
mesh_terms: D058917:Adenosine A2 Receptor Antagonists / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D005901:Glaucoma / Q000188:drug therapy; D006874:Hydrostatic Pressure / Q000009:adverse effects*; D007249:Inflammation / Q000150:complications* / Q000503:physiopathology; D008070:Lipopolysaccharides / Q000494:pharmacology; D023421:Models, Animal; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D009924:Organ Culture Techniques; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D014230:Triazoles / Q000494:pharmacology
chemical_list: C098657:5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine; D058917:Adenosine A2 Receptor Antagonists; D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D011743:Pyrimidines; D014230:Triazoles; D009569:Nitric Oxide