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Makoto Ishikawa, Takeshi Yoshitomi, Charles F Zorumski, Yukitoshi Izumi; Synergistic protection from high pressure-induced injury by allopregnanolone and 24(S)-hydroxycholesterol in the ex vivo rat retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4903.
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© ARVO (1962-2015); The Authors (2016-present)
Allopregnanolone (AlloP) and 24(S)-hydroxycholesterol (24SH) are neurosteroids (NS) synthesized in the retina that modulate GABA and glutamate receptors, respectively. We recently reported that these NS are neuroprotective against glaucomatous damage when administered separately at 1 μM. In the present study, we examined whether co-administration of lower concentrations of AlloP (100 nM) and 24SH (100 nM) protect retinal ganglion cells (RGCs) more effectively from intraocular pressure elevation.
Rat ex vivo eyecups were prepared from 30-day-old male Sprague-Dawley rats. Eyecups were incubated in artificial cerebrospinal fluid (aCSF) 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. AlloP (100 nM) and 24SH (100 nM) were separately or simultaneously administered into the aCSF (n=5 for each experiment). Tissue was also processed to determine RGC injury using anti-NeuN antibody (a RGC nuclear marker) and TUNEL staining (n=5 for each experiment). For comparison with controls, we used Dunnett's multiple comparison test.
In whole mounted retinas, the labeling density of anti-NeuN antibody in the RGC (LDN) was 1579.0/mm2 at 75 mmHg (control). Neither 100 nM AlloP alone (LDN=1976.7/mm2, p>0.05) nor 100 nM 24SH alone produced significant RGC protection (LDN=1918.1/mm2, p>0.05), while co-administration of 100 nM AlloP and 100 nM 24SH significantly promoted survival of NeuN-positive RGCs at 75 mmHg (LDN=2497.0/mm2, p<0.05) compared to control. Exposure to elevated pressure induced apoptosis in the GCL and INL (LDA=33.6/200 μm, control). Again, neither 100 nM AlloP alone (LDA=14.2/200 μm, p>0.05) nor 100 nM 24SH alone (LDA=13.2/200 μm, p>0.05) produced significant protection, while co-administration of 100 nM AlloP and 100 nM 24SH substantially inhibited apoptosis in the retina at 75 mmHg (LDA=0.4/200 μm, p<0.05) compared to control.
Co-administration of AlloP and 24SH may serve as effective therapeutic strategies for protecting glaucomatous eyes from pressure-induced injuries.
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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