Abstract
Purpose:
Histone deacetylase (HDAC) inhibitors have been associated with potential neuroprotective properties in relation to glaucoma. The purpose of these experiments was to investigate the effect of the HDAC inhibitor trichostatin A (TSA) on gene expression in human organotypic retinal cultures (HORCs).
Methods:
Donor eyes were obtained from the East Anglian Eye Bank within 24 hours post mortem. 4mm diameter paramacular explants were dissected from the retina and cultured in serum free DMEM/Ham F12 medium for 48 hours in the presence or absence of TSA (0.1, 1 or 10µM). LDH release was used to assess cell death. Total RNA levels were assessed by spectrophotometric analysis. Gene expression was assessed by QRT-PCR.
Results:
There was no significant change in LDH release from the HORCs following 48 hours' exposure to 0.1 - 10µM TSA indicating that the HDAC inhibitor was not causing toxicity (n=4). During the culture period, there was an approximate 60% decrease in total RNA (n=4). TSA led to a significant amelioration of loss of total RNA (approximately 25% at 10μM; n=4). Analysis of the expression of two housekeeping genes (cytochrome c-1; CYC1 and topoisomerase 1; TOP1) showed no significant change in expression in TSA-treated retina compared with control. The expression of three markers for retinal neurons was also assessed: Thy-1 (THY-1) for retinal ganglion cells (RGCs); recoverin (RCVRN) for photoreceptors and calbindin (CALB) for horizontal cells. There was no significant change in expression of CALB in HORCs treated with TSA compared with control (n=4). RCVRN showed a significant increase in expression of approximately 20% (n=4) and THY-1 an approximate 80% increase (n=4) in expression in HORCs treated with 10µM TSA compared with control at the 48 hour time point.
Conclusions:
HDAC inhibition with TSA differentially inhibited loss of gene expression in the cultured human retina. Of the genes investigated, the RGC marker THY-1 showed the greatest protection. Loss of normal gene expression has been shown to be an early event in animal models of glaucoma. The current data may support a potential neuroprotective role for HDAC inhibitors in relation to glaucoma.
Keywords: 694 retinal culture •
615 neuroprotection •
531 ganglion cells