An Empty E1–, E3–, E4– Adenoviral Vector Ameliorates Light–Induced Photoreceptor Degeneration

Abstract

Abstract: : Purpose: Previously, we reported that adenoviral vector delivery of pigment epithelium–derived factor (AdPEDF) rescued photoreceptors from light–induced cell death (Imai, et al., 2004). We also found that empty vector had some neuroprotective effect though significantly less than AdPEDF. The purpose of this study is to better understand and evaluate the neuroprotective effect associated with empty E1^–, E3^–, E4^– adenoviral vector treatment, in a murine model of light–induced, photoreceptor degeneration. Methods: Dark–adapted BALB/c mice, aged 6–8 weeks, were exposed to standardized, intense fluorescent light for 12, 96 or 144 hours. Prior to dark–adaptation all mice received intravitreous injection of 1x10⁹ particles of an empty E1^–, E3^–, E4^– adenoviral vector in one eye and vehicle in the other. Following light challenge of 96 and 144 hours, histopathological analysis, including quantitative photoreceptor cell counts (number of photoreceptors per 50 µm), was conducted. Semi–quantitative assessment of mRNA for the apoptosis related genes: p50, p65, IkBa, caspase–1, caspase–3, Bad, Jun, Bax, Bak, Bcl–2, fos and p53 using qRT–PCR was performed on eyes following 12 hours light exposure. Results: After 96 hours of light exposure photoreceptor cell density for E1^–, E3^–, E4^– adenoviral vector and vehicle–injected eyes were 87.5 ± 9.5 and 79.3 ± 9.0, respectively, (p=0.14). After 144 hours light exposure photoreceptor cell density was preserved in vector–injected eyes as compared to vehicle treated eyes, 68.9 ± 9.9 and 49.2 ± 4.1, respectively, (p=0.016). Relative mRNA levels of p50, caspase–1 and Bcl–2 differed significantly between vector– and vehicle–injected eyes (p=0.026, 0.034, 0.028, respectively). The other apoptosis related genes evaluated were not significantly different. Conclusions: These data indicate that intravitreous injection of empty E1^–, E3^–, E4^– adenoviral vector increases photoreceptor cell survival resulting from intense light exposure. Associated gene expression changes suggest that the protective effect involves the NFkB/caspase–1 pathway as well as an anti–apoptotic effect of Bcl–2. These studies will assist in the identification of other potential therapeutic neuroprotective genes that can be used in conjunction with PEDF.

Keywords: adenovirus • apoptosis/cell death • gene transfer/gene therapy