title: NF-κB-mediated nitric oxide production and activation of caspase-3 cause retinal ganglion cell death in the hypoxic neonatal retina.
abstract: OBJECTIVE
  Hypoxic insult to the developing retina results in apoptosis of retinal ganglion cells (RGCs) through production of inflammatory mediators, nitric oxide (NO), and free radicals. The present study was aimed at elucidating the pathway through which hypoxia results in overproduction of NO in the immature retina, and its role in causing apoptosis of RGCs.
  
  
  METHODS
  Wistar rats (1 day old) were exposed to hypoxia and their retinas were studied at 3 hours to 14 days after exposure. The protein expression of nuclear factor-κB (NF-κB) and neuronal nitric oxide synthase (nNOS) in the retina and primary cultures of RGCs was analyzed using Western blotting and double-immunofluorescence, whereas the concentration of NO was determined calorimetrically. In cultured RGCs, hypoxia-induced apoptosis was evaluated by caspase-3 immunolabeling.
  
  
  RESULTS
  Following hypoxic exposure, NF-κB-mediated expression of nNOS, which was localized to the RGCs, and subsequent NO production was significantly increased in the developing retina. In primary cultures of RGCs subjected to hypoxia, the upregulation of nNOS and NO was significantly suppressed when treated with 7-nitroindazole (7-NINA), an nNOS inhibitor or BAY, an NF-κB inhibitor. Hypoxia-induced apoptosis of RGCs, which was evident with caspase-3 labeling, also was suppressed when these cells were treated with 7-NINA or BAY.
  
  
  CONCLUSIONS
  Our results suggest that in RGCs, hypoxic induction of nNOS is mediated by NF-κB and the resulting increased release of NO by RGCs causes their apoptosis through caspase-3 activation. It is speculated that targeting nNOS could be a potential neuroprotective strategy against hypoxia-induced RGCs death in the developing retina.
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Gurugirijha
authors.initials: G
authors.lastname: Rathnasamy
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Sivakumar
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Parakalan
authors.initials: P
authors.lastname: Rangarajan
authors.affiliation: Singapore Eye Research Institute, Singapore National Eye Centre, Singapore Emeritus Professor, University of Glasgow, Glasgow, Scotland.
authors.forename: Wallace S
authors.initials: WS
authors.lastname: Foulds
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Eng Ang
authors.initials: EA
authors.lastname: Ling
authors.affiliation: Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Singapore Eye Research Institute, Singapore National Eye Centre, Singapore.
authors.forename: Charanjit
authors.initials: C
authors.lastname: Kaur
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000502:physiology*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D000860:Hypoxia / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012164:Retinal Diseases / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000378:metabolism*
chemical_list: D016328:NF-kappa B; D009569:Nitric Oxide; D053148:Caspase 3
keywords: 7-nitroindazole; NF-κB; caspase-3; hypoxia; neuronal nitric oxide synthase; nitric oxide; retinal ganglion cells