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Jens Nääv Ottosson, Linnea Taylor, Karin Arner, Fredrik K Ghosh; Acetazolamide and inner retinal fluid homeostasis: gliotic changes in cultured porcine retina. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1747.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effects of acetazolamide (AZ), a carbonic anhydrase inhibitor used to treat glaucoma, on protein expression related to retinal fluid homeostasis, gliosis and apoptosis in adult porcine retinal explants.
Retinal explants were cultured using a standardized protocol, with and without AZ added to the medium, for two and five days. Specimens were fixed, cryosectioned and stained with hematoxylin and eosin as well as immunohistochemical markers for Müller cells and photoreceptors. Apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Fluorescence intensity was analyzed using ImageJ, and acquired data was processed using an ANOVA with a Tukey post hoc test. Eyes fixed immediately after enucleation were used as in vivo controls.
Hallmarks of retinal gliosis and neuronal degeneration were seen in all cultured explants, especially in the 5 DIV specimens, including: an upregulation of glial fibrillary acidic protein (GFAP) and a decrease in the number of ganglion cells. A reduced number of ganglion cells was seen in 5DIV specimens treated with AZ compared to 5DIV controls, although this difference was not statistically significant. Apoptosis was significantly increased in AZ 5DIV compared to 5DIV controls (P < 0,05). All cultured specimens displayed an increased expression of Kir4.1, and there was a reduced expression of Kir4.1 in the 5 DIV specimens treated with AZ compared to their untreated counterparts (P < 0,01). Significant loss of polarization in cultured retinas was observed in Kir4.1 as well as aquaporin-4 (AQP4) labeling, although the total expression of AQP4 remained unaltered. In contrast with carbonic anhydrase II (CAII), which revealed no differences in expression, carbonic anhydrase XIV (CAXIV) was upregulated in all cultured specimens.
All cultured explants displayed clear signs of gliosis, as well as an upregulation of Kir4.1 and CAXIV and a loss of polarity in the expression of Kir4.1 and AQP4. In the 5DIV specimens treated with AZ, there was a downregulation of Kir4.1 and an increase in the number of apoptotic cells compared with untreated counterparts, as well as a trend towards increased ganglion cell death. Considering the frequent use of AZ in the treatment of glaucoma, these results merit further investigation.
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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