title: The development of human organotypic retinal cultures (HORCs) to study retinal neurodegeneration.
abstract: OBJECTIVE
  To develop human organotypic retinal cultures (HORCs) to study retinal ganglion cell (RGC) death in response to ischaemic and excitotoxic insults, both known to cause loss of RGCs and proposed as mechanisms involved in glaucomatous retinal neurodegeneration.
  
  
  METHODS
  Human donor eyes were obtained within 24 h post mortem. The retina was isolated and explants cultured using two techniques. THY-1 mRNA (assessed by real-time quantitative PCR) and neuronal nuclei (NeuN) (assessed by immunohistochemistry) were used as markers of RGCs. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL).
  
  
  RESULTS
  The distribution of THY-1 mRNA and NeuN-labelling within the human retina was consistent with the expected distribution of RGCs. Gross morphology and retinal architecture remained stable over a 96 h culture period. THY-1 mRNA and NeuN-labelled RGC layer cells decreased over the culture period, and there was an increase in TUNEL-labelling with time, but HORCs cultured in serum-free DMEM/HamF12 medium were useful for up to 48 h in culture. N-methyl-d-aspartate (10 μM) caused a reduction in THY-1 mRNA by 24 h and decreased the numbers of NeuN-labelled RGC layer neurons by 48 h, suggesting that the loss of THY-1 mRNA was a marker of RGC stress prior to death. Simulated ischaemia (60 min oxygen/glucose deprivation) caused a reduction at 24 h in both THY-1 mRNA and the numbers of NeuN-labelled neurons of HORCs.
  
  
  CONCLUSIONS
  HORCs provide a useful model to investigate RGC insult by neurodegenerative mechanisms that may lead to glaucoma in human eyes.
authors.affiliation: Department of Ophthalmology, Norfolk and Norwich University Hospital, Norwich, UK.
authors.forename: Nuwan
authors.initials: N
authors.lastname: Niyadurupola
authors.forename: Peter
authors.initials: P
authors.lastname: Sidaway
authors.forename: Andrew
authors.initials: A
authors.lastname: Osborne
authors.forename: David C
authors.initials: DC
authors.lastname: Broadway
authors.forename: Julie
authors.initials: J
authors.lastname: Sanderson
mesh_terms: D017209:Apoptosis; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D005901:Glaucoma / Q000209:etiology; D006801:Humans; D007150:Immunohistochemistry; D016202:N-Methylaspartate / Q000235:genetics; D009410:Nerve Degeneration / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism*; D012162:Retinal Degeneration / Q000473:pathology*; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000235:genetics / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*; D014795:Visual Pathways
chemical_list: D012333:RNA, Messenger; D018800:Thy-1 Antigens; D016202:N-Methylaspartate