Abstract
Purpose: :
Neuronal injury and cell loss are often changes noted in neuro-ophthalmic diseases on the optic nerve exam. No therapies exist to date to protect or revive these damaged structures. Evidence is accumulating that the heat shock response (hsr) is neuroprotective against a wide variety of insults both in vitro and in vivo. Some of these agents may be neuro-regenerative as well. The goal of our current studies was to determine if stimulating the heat shock response has a neuroprotective effect on retinal ganglion cells (RGCs).
Methods: :
A well-established rat retinal ganglion cell line (RGC-5) was cultured by previously described methods. Cells were treated with varying concentrations of heat shock inducers, Celatsrol and Geldanamycin, for 24 hours and the optimum dose was determined based on measuring cellular HSP70 levels by western blot. Doxorubicin, a known oxidative apoptotic agent, was added to cells at various concentrations. Cells were harvested and viability was determined after trypan blue staining using a Cellometer (Nexcelcom Biosciences). RGC-5 cells were then pre-incubated with celastrol (CSA) or geldanamycin (GA) for 24 hours. Thereafter, cells were exposed to doxorubicin and at various times post-insult the number of viable cells was quantitated.
Results: :
We first studied the dose and time-dependent nature of RGC cell death in the presence of doxorubicin. Cells were treated with Doxorubucin concentration ranging from 0.5 to 5 ug/ml. About 60-80% cell death was observed with the higher doses of Doxorubicin after 24 hours incubation. However pre-treatment with either CSA or GA there was a statistically significant reduction in cell death to about 20%. Analysis of doxorubicin exposed cells treated with the placebo (DMSO) showed profound morphological changes associated with apoptosis. Additionally, IF revealed that either CSA or GA-treated cells showed minimal apoptotic features.
Conclusions: :
For the first time, we demonstrate that stimulating the heat shock response in RGC’s provides neuroprotection against doxorubicin-induced apoptosis. There are a variety of chemicals that stimulate the heat shock response in different ways. These compounds may clinically provide neuroprotection to the retinal ganglion cells, allowing us to mitigate RGC loss in such diseases as non-arteritic anterior ischemic optic neuropathy, glaucoma and mitochondrial diseases.
Keywords: neuroprotection • apoptosis/cell death • neuro-ophthalmology: optic nerve