Circadian Effects on Retinal Morphology: Relevance to Gene Transfer

Abstract

Abstract: : Purpose:While performing morphological studies for effects of retinal gene therapy, we observed significant alterations in the size and shape of photoreceptor components depending on the stage of the light cycle. The objective of this study is was to further characterize circadian morphological changes in photoreceptors and then to evaluate cellular transduction characteristics of an adenoviral vector at different circadian stages. Methods: CD–1 mice were housed under light–dark (LD, 12/12 hr) cycles and eyes were harvested from cohorts of animals at 4 different times (at 6 hour intervals) relative to the time of light onset (defined as zeitgeber, ZT). Lengths of outer segments (OS), inner segments (IS) and number of nuclei in the outer nuclear layer (ONL) were measured in frozen and plastic sections. Immunohistochemistry was used to label photoreceptor components. Additional cohorts of mice were injected unilaterally and subretinally with Ad.LacZ at ZT–6 and ZT–18. Three days after injection, the eyes were enucleated and processed for frozen and plastic sections. LacZ expression was monitored through X–Gal histochemistry. Results:The outer and inner segments displayed maximal elongation at ZT 6 and minimal length at ZT 18. Immunolabeling revealed dramatic circadian effects on localization and levels of several molecules including rhodopsin and vascular endothelial growth factor (VEGF). Efficient photoreceptor transduction was noted after subretinal injection of Ad.LacZ at ZT 18, when outer segments were shortest, but not at ZT 6. RPE cells were targeted efficiently regardless of time of injection. Conclusions: Photoreceptors undergo dramatic morphological changes based on circadian rhythm. We have harnessed this information to increase photoreceptor transduction efficiency of a viral vector which does not normally target differentiated photoreceptors.

Keywords: photoreceptors • gene transfer/gene therapy • microscopy: light/fluorescence/immunohistochemistry