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Heather M Schmitt, Cassandra Schlamp, Robert W Nickells; Systemic dosing of an HDAC3-selective inhibitor prevents retinal ganglion cell nuclear atrophy and apoptosis after optic nerve injury. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2554. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Histone deacetylases (HDACs) regulate nuclear atrophy early after axonal injury to retinal ganglion cells (RGCs). Specifically, HDAC3 plays an important role in histone deacetylation, heterochromatin formation, and apoptosis in RGCs post optic nerve crush (ONC). Conditional knockout of Hdac3 prior to ONC and intravitreal injection with RGFP966, an HDAC3 selective inhibitor, immediately after ONC both prevent nuclear atrophy in RGCs. However, repeated intravitreal dosing of RGFP966 is not feasible to treat chronic loss of RGCs. Therefore, validation of a therapeutic effect after systemic dosing of RGFP966 is necessary.
C57BL/6 mice were injected intraperitoneally (IP) with 10mg/kg of RGFP966, sacrificed at 0, 0.5, 1, 2, 6, and 24 hours post injection, and retinas were harvested to measure bioavailability of RGFP966 using mass spectrometry. Mice were also injected IP with RGFP966 doses ranging from 2mg/kg-10mg/kg immediately following ONC. Eyes were then harvested at 5 days post ONC to measure acetylated histone H4 (AcH4) and BRN3A positive cells in the ganglion cell layer (GCL) using immunofluorescence. Mice were injected IP with 10mg/kg RGFP966 daily, every 3 days, and every 7 days following ONC surgery, and total cell counts were obtained on retinas at 14 days post ONC. H&E staining was used to analyze the pathology of organ tissues such as the brain, heart, lungs, and small intestine after 14 days of daily systemic injection of RGFP966.
RGFP966 crossed the blood-retinal barrier, with a peak concentration of 15mM per retina at 1 hour after a 10mg/kg IP injection. A single IP injection of RGFP966 ranging from 2mg/kg-10mg/kg, given immediately after ONC, significantly prevented histone deacetylation at all doses (p<.0005). Daily repeated IP injections of 10mg/kg RGFP966 over the course of 2 weeks post ONC prevented RGC loss. Preliminary H&E staining results indicate no toxic effects to off-target tissues in mice treated daily for 14 days. Experiments on RGFP966 dosing in chronic models of experimental glaucoma are also in progress.
Preliminary experiments indicate that inhibition of HDAC3 activity with systemic dosing of RGFP966 prevents histone deacetylation and eventual apoptosis of RGCs post acute optic nerve injury.
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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