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S. Zhang, J. Wu, X. Sun; Valproic Acid Enhances Transduction and Promotes Retinal Ganglion Cell Survival Mediated by rAAV2-BDNF Following Optic Nerve Transection. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2757.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effects of Valproic acid (VPA), a potent Histone deacetylase inhibitor (HDACi), on the gene expression and neuroprotection mediated by rAAV2 following optic nerve transection.
The rAAV2-EGFP/rAAV2-Luc alone or with lower dosage of VPA was intravitreally injected into either SD rats or Balb/c mice. EGFP expression in the living retina was monitored and photographed using fluorescent stereoscope. Bioluminescence imaging was performed to evaluate dynamics of transgene expression in vivo. This strategy, the combination of rAAV2 and VPA, was further investigated for the feasibility to augment the neuroprotective effects following optic nerve transection of SD rat. rAAV2-BDNF combined with or without VPA was intravitreally injected immediately after optic nerve transection in SD rats. Retinal ganglion cells (RGCs) survival was quantified by the counting of retrograde Dil-labeled RGCs in flat-mounted retinas at 1, 2 and 4 weeks after surgery. The protein expressions of transgene EGFP and BDNF in the treated retinas were detected by western blotting. The mRNA expression and DNA copy number of transgene EGFP were determined at the 1, 3, 5, and 7 days after injection using real-time PCR.
Earlier onset of gene expression, stronger and prolonged fluorescence were observed in the rAAV2 with VPA injected eyes in comparison with rAAV2 injected eyes. The enhancement of transgene expression reached a plateau 4 weeks after injection, and then up to 8 weeks postinjection. In the optic nerve transection models, significantly more surviving RGCs were observed in the rats received injection of rAAV2-BDNF and VPA than in the rats injected with rAAV2-BDNF alone. Only 21.2% ± 2.4% RGCs were lost 2 weeks after injury in the retinas injected with rAAV2-BDNF and VPA, whereas the control, received injection of rAAV2-BDNF alone, lost 37.6% ± 3.1% of RGCs (p<0.01). Western blot analysis confirmed that VPA resulted in a robust dose-and time-dependent increase of rAAV2-mediated gene expression in the retinas in vivo. Real-time PCR analysis shown that combination of VPA produced a more than 6-fold increase in EGFP mRNA levels (at 5 days), compared to infection with rAAV2-EGFP alone. However, VPA did not significantly affect the copy number of the rAAV2.
The data from this study suggested that VPA could exert a significant enhancing and accelerating effect on rAAV2-mediated gene expression in retinal cells both in rat and mouse. The combinatorial use of VPA and rAAV2-BDNF produced improvement of RGCs survival after optic nerve transection.
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