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maryam alavi, Petr Baranov; Characterize growth factor expression in the developing and aging mouse retina in vivo and in vitro. Invest. Ophthalmol. Vis. Sci. 2017;58(8):603.
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© ARVO (1962-2015); The Authors (2016-present)
It has been shown that growth factor administration or induction in the can delay or prevent retinal neuron death. However, the changes in the retinal secretome in development, aging and disease have not been well described. In this study we analyzed the expression of therapeutically relevant growth factors, GDNF, PEDF, CNTF, bFGF in in the developing and aging mouse retina in vivo and in vitro.
To estimate the growth factor content in the retina, mouse eyes (3 months and 2 years old, C57Bl6/J, n=7 per group) were collected. The neural retina with the RPE was separated from the underlying tissues, lysed and processed for the RNA and protein isolation. Western Blotting and qPCR was used to assess mRNA and protein concentrations of growth factors (GDNF, PEDF, CNTF, bFGF) in the retina; bActin and GAPDH were used as internal controls. In addition, immunostaining assay was used to localize growth factor localization. To study the GF in the developing retina we used mouse iPSc differentiation to mimic retinal development in vitro. The supernatant from the cultures of ipsc-derived eyecups was collected at several stages of differentiation (Days 0, 5, 11, 16, 20, 25, 30, 35). The growth factor content (PEDF, GDNF) was analyzed using ELISA (RnD Systems). We used t-test and one-way ANOVA for the statistical analysis.
The absolute content of GDNF in the eye decreased with age from 9.19 ± 3.36 ng per retina to 5.80 ± 3.47 ng per retina (n=10, p-value=0.03; The result is significant at p < 0.05). The regional analysis showed no difference in the GDNF expression in the choroid, neural retina, iris and vitreous (p-value =0.706). The in vitro study demonstrated gradual increase in GDNF and PEDF expression in culture from 1pg/ml to 8pg/ml for GDNF and from 80pg/ml to 166pg/ml for PEDF.
We have observed a decrease in GDNF expression with aging, which may be responsible for previously described age-related changes in the healthy mouse retina. The GDNF content in the retina, identified in our work, can be used as a benchmark for the GDNF delivery/induction studies. We also have demonstrated the utility of the three-dimensional retinal differentiation from the iPSc in secretome studies. This assay may be used to investigate the modulation of growth factor expression in the retina.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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