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Pedro Gonzalez, Dana M Garcia; Optic nerve astrocytes of zebrafish (Danio rerio) as a model for aging studies in humans. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5064.
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© ARVO (1962-2015); The Authors (2016-present)
Astrocytes, involved in maintaining neuronal health within the central nervous system, express glial fibrillary acidic protein (GFAP) at high levels in response to trauma or disease. A considerable amount of research is directed at the changes astrocytes undergo over time to better understand the process of aging. Using confocal microscopy, we tested the hypothesis that increased expression of GFAP correlates with increased age using the optic nerve (ON) of zebrafish as an experimental model.
Zebrafish (D. rerio) ages 3, 6, 9 and 12 months of age were purchased from the Zebrafish International Resource Center (ZIRC). Four fish from each age group were tested to determine if there was a trend in expression of gfap. Fish were euthanized and placed in 4% paraformaldehyde solution overnight. Fish were then measured and dissected to obtain the eyes and brain. The tissue was then frozen, embedded and sectioned into 20-μm thick sections using a Shandon cryotome and afterwards adhered to gelatin-coated coverslips. Antibodies for immunolabeling Gfap were anti-GFAP (zrf-1) raised in mice (1:200 dilution) as the 1° antibody and goat anti-mouse Alexa Fluor 488 (1:300 dilution) as the 2° antibody. Once labeling was complete, positive and negative control images were acquired using an Olympus FV1000 confocal laser-scanning microscope.
Our results indicate absence of Gfap labeling in the optic nerve of 3-month-old zebrafish. In contrast, the 6, 9, and 12-month-old zebrafish show presence of Gfap in the optic nerve, but no notable differences in the expression levels are evident in this age range. Figure A depicts the optic nerve of a 3-month-old zebrafish and B depicts the optic nerve of a 12-month-old zebrafish both using a 60x oil immersion lens. The bright green color indicates Gfap labeling and blue color labels for nuclei.
With the important caveat that the range of ages we examined does not include older fish, our hypothesis is not supported by these data; however, further experiments are being conducted to quantify the data, as well as to examine older fish (24, 36, 48-month-olds). Additionally, experiments are currently underway to determine if Gfap expression correlates with markers for senescence, such as p16. If both of these indicators for aging and senescence are observed, then zebrafish may be a defensible animal model for understanding aging in humans.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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