Abstract
Purpose: :
To investigate the long term survival of retinal ganglion cells (RGCs) in human organotypic retinal cultures (HORCs) maintained for extended periods of up to 4 weeks.
Methods: :
Donor eyes were obtained from the East Anglian Eye Bank within 24hours post mortem. The retina was dissected and five retinal explants (4mm in diameter) were taken from the paramacular regions of the retina. The HORCs were cultured for up to 4 weeks in DMEM/HamF12 +/- FCS or Neurobasal medium supplemented with B27N2. RGC numbers were assessed by NeuN immunohistochemistry and level of THY-1 expression (QRT-PCR). Apoptosis was evaluated by TUNEL assay.
Results: :
Under all conditions basic retinal morphology and architecture were preserved, with the nuclear layers remaining well defined over the 4 week culture period. In all conditions there was a significant decrease in the number of NeuN-labelled RGC s over the culture period, with the greatest loss (approximately 60% at 4 weeks) being seen in Neurobasal medium (n=4; p<0.05). There was a corresponding increase in the number of TUNEL-positive NeuN-labelled cells under all conditions, again with culture in Neurobasal medium resulting in the highest proportion of apoptotic nuclei (almost 100%). DMEM/HamF12 supplemented with serum showed the best survival with no significant loss of NeuN-labelled cells until the 3 week time point (n=4; p<0.05). Under all conditions, THY-1 expression declined significantly by week 1, then remained at a baseline level of approximately 20% for the 4 week culture period (n=4; p<0.05).
Conclusions: :
There was a decline in the number of RGCs in human organotypic retinal cultures in long-term culture. Loss of RGCs in Neurobasal medium with B27/N2 supplementation was greater than in SF DMEM/HamF12. Loss of RGCs could be protected by serum supplementation. This long-term culture model might be useful for investigation of neurodegeneration and neuroprotection of human RGCs.
Keywords: retinal culture • neuroprotection • ganglion cells