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Ray A. Enke, Karl J. Wahlin, Verity F. Oliver, Mariam S. Assadian, Kieron Torres, Donald J. Zack, Shannath L. Merbs; Characterizing the Role of Epigenetic Mechanisms in Apoptotic Retinal Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5983.
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Retinal cells undergo programmed cell death (PCD) during normal development as well as in response to injury and disease. We hypothesize that epigenetic mechanisms may contribute to apoptosis of retinal neurons in the developing and diseased retina. The role of epigenetic modifications, such as DNA methylation, in public health and medicine has primarily concentrated on the malignant transformation of cells. A more recent focus has been the possible contribution of epigenetic changes to tissue development and tissue-specific diseases. To determine a correlation between epigenetic mechanisms and PCD in the retina, we used immunohistochemical (IHC) analysis of the epigenetic modification 5-methyl cytosine (5-meC) in experimental models of retinal disease and development.
Retinal cross sections were prepared from chicken at different time points during development. Retinal sections were also prepared from adult mouse models of retinal disease, the rd1 model of retinal degeneration and the DBA/2J model of glaucoma. Eye cups from adult animals or heads from embryonic animals were fixed in 4% PFA and cryoprotected. Retinal cross sections were cut and used for IHC analysis using an antibody against 5-meC as well as markers of PCD.
TUNEL positive neurons in the degenerating retina of adult rd1 mice demonstrate enhanced staining for 5-meC. This was also observed in a developmental time course of chicken retinas. Although cleaved caspase-3 (cCaspase-3) positive retinal neurons were present at similar periods of development there was little overlap with 5-meC positive cells suggesting that they are expressed at different stages of PCD. We are currently evaluating the 5-meC IHC staining of the DBA/2J mouse glaucoma model to determine if there is a similar correlation in retinal ganglion cells undergoing apoptosis, and these results will also be presented.
The genomes of retinal neurons in the late stages of PCD demonstrate DNA hypermethylation by IHC. This finding suggests that, in vertebrates, epigenetic mechanisms may play a role in apoptosis of neurons during retinal development and retinal degeneration. Further investigation is needed to determine if other canonical epigenetic marks correlate with hypermethylation in apoptotic neural retina.
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