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Makoto Ishikawa, Takeshi Yoshitomi, Douglas Covey, Charles Zorumski, Yukitoshi Izumi; TSPO modulates retinal axonal swelling in a rat ex vivo glaucoma model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2572.
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© 2017 Association for Research in Vision and Ophthalmology.
Allopregnanolone (AlloP) is a neurosteroid and exerts neuroprotective effects against glaucomatous pressure-induced injuries, although mechanisms underlying neurosteroidogenesis have not yet been clarified. The aim of this study was to determine whether AlloP synthesis is induced by activation of translocator protein 18 kD (TSPO) and whether TSPO modulates retinal axonal swelling in an ex vivo glaucoma model.
Rat ex vivo eyecups were prepared from 28-32 day old male Sprague-Dawley rats. Eyecups (n=20) were placed at the bottom of 100 ml glass beakers filled with artificial cerebrospinal fluid (aCSF), and incubated at 30°C for 24 hours using a closed pressure system. The pressure in the chamber was increased by introducing 95% O2 and 5% CO2 through an infusion valve. After maintaining the chamber at a set pressure (10 or 75 mmHg) for the indicated time, the pressure inside the chamber was carefully decreased (n=15 at each pressure). In some experiments, TSPO antagonist (atriol, 1 μM, n=14) and partial agonist (PK11195 (PK), 50 μM, n=15) were administered in aCSF at 75 mmHg. AlloP concentration was measured by LC-MS/MS (n=4 to 5 for each experiment). Tissue was also processed for immunochemistry and light microscopy (n= 5 for each experiment). Two-tailed Student’s t-test was used for statistical analysis.
In this acute model, LC-MS/MS analyses revealed that AlloP production significantly increased at 75 mmHg (61.1±11.08 ng/g, p=0.008) compared to control pressure (10 mmHg, 1.78±0.37 ng/g). At 75 mmHg, administration of PK markedly increased AlloP (116.2±36.96 ng/g) compared to control group incubated without PK (p=0.0067). Conversely, enhanced AlloP expression at 75 mmHg was substantially blocked by atriol (7.33±1.43 ng/g, p=0.014). Immunochemistry revealed an increase in expression of AlloP and TSPO in the RGC and INL at 75 mmHg compared to 10 mmHg. As previously described, pressure elevation (75 mmHg) induced axonal swelling in the NFL (12.9±3.4 μm). Administration of PK remarkably diminished pressure-mediated axonal swelling at 75 mmHg (2.1±1.4 μm, p=0.0002), while atriol overcame these neuroprotective effects (10.1±2.3 μm, p=0.16).
AlloP synthesis is modulated by activation of TSPO in an ex vivo glaucoma model. TSPO agonists may serve as potential therapeutic targets for the prevention of pressure-induced retinal damage in glaucoma.
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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