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Petr Y Baranov, Tatiana Sukhanova, Oleg Yerov, Hong Lin, Chris James, Dwight Morrow, Shaohui Wang, Phoebe Lei, John McNeish, Michael J Young; Amitriptyline induces glial-cell line derived neurotrophic factor in retinal cells in vivo and in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5744.
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Retinal degenerative disorders, such as age-related macular degeneration and retinitis pigmentosa, cause progressive loss of photoreceptors. In pre-clinical models of retinal dystrophy, treatment with growth and neurotrophic factors including GDNF, CNTF, PEDF and NGF results in substantial protection and reduction of neuronal loss over time when administered either by direct injection, use of controlled release systems or gene transfer using AAV or plasmids. Each route of administration, however, suffers from at least one major drawback To establish a screen using retinal-derived cells to identify small molecules that can induce the production of GDNF. The major objective is to identify drugs that induce GDNF that can be administered directly into the eye.
The cell lines chosen for these studies included human retinal progenitor cells (hRPC), an immortalized, human retinal pigment epithelial cell line (ARPE19), and a transformed human astroglia (SVG) cell line. We tested several low-molecular weight compounds that have been shown to induce GDNF in rat C6 glioma cells: valproic acid, amitriptyline, cyclobenzaprine, amoxapine, and loxapine. To study the effect of amitriptyline in vivo we performed intravitreal injections of 10uM amitriptyline solution into albino RCS rats. Total RNA from neural retina and RPE was isolated 2 hours after injection, and protein 6 hours after injection. The level of GDNF expression was analyzed by RT-PCR, protein - by WB.
Amitriptyline, Cyclobenzaprine, and Loxapine treatment showed dose-dependent upregulation of GDNF protein in all cell types tested though the amount of GDNF induction varied (ELISA). In contrast, valproic acid showed robust induction only in APRE19 & SVG, but not hRPC. Flow cytometry results were consistent with ELISA. RT-PCR showed the upregulation of GDNF mRNA (1.6 fold) and protein (1.8 fold) in RCS rat retinas following amitriptyline injection.
We are developing an assay to screen for drugs that induce GDNF in retinal cells. We found that several molecules known to induce GDNF in C6 glioma cells also induce GDNF in ARPE-19 and hRPCs, and amitriptyline has a similar effect in vivo. This approach will enable identification of drugs that can be used to induce GDNF expression and serve as a neuroprotective agent in retinal degenerative disorders.
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