Abstract
Purpose :
Retinal ischemia is important in the pathogenesis of glaucoma and diabetic retinopathy. In glaucoma, retinal ischemia blocks axonal transport, depletes neurotrophic factors, degenerates the optic nerve, and causes loss of retinal ganglion cells (RGCs). Such changes can lead to visual impairment and irreversible blindness. FK962 stimulates the release of neurotrophic factors in the brain. Our previous study in cultured rat RGCs showed that FK962 induced neurite elongation. However, the mechanism for the neuroprotective effect of FK962 was not established. The purpose of the present experiments was to determine the mechanism for FK962 amelioration of retinal neuronal cell death.
Methods :
Rat retinal cells (neuronal and glial cells) were cultured for 48 hrs in supplemented medium and then received: 1) 24 hrs of hypoxia, or 2) 24 hrs of hypoxia plus 24 hrs of reoxygenation in medium without supplements. FK962 or a combination of BDNF, CNTF, and forskolin (NFs) were presented 2 hrs before and during hypoxia/reoxygenation. Cells were identified as neuronal cells and RGCs if they immunostained positive for neurofilament H (NF-H) and double positive for NF-H and Brn3a, respectively. Propidium iodide (PI) stained for necrosis and/or late stage apoptosis. Activation of caspases 3/7, markers for presumed apoptosis, was detected using fluorescent-labeled substrates.
Results :
Even under normoxia, neuronal cells and RGCs decreased in a time dependent manner due to depletion of supplements in the medium. Hypoxia/reoxygenation caused further, significant decreases in cell numbers. FK962 and NFs significantly ameliorated this cell loss. In neuronal cells cultured under hypoxia/reoxygenation, large numbers of PI-positive and caspase-positive neurons were observed, suggesting involvement of both necrosis and apoptosis in hypoxic cell death. FK962 and NFs significantly reduced necrotic and apoptotic neuronal cells. Interestingly, all surviving neurons expressed developed neurites regardless of culture conditions, and increased numbers of these more mature surviving cells were found in the FK962- and NFs-treated groups.
Conclusions :
FK962 may prevent necrosis and apoptosis by allowing persistence of more mature retinal neurons.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.