A Novel, Real-Time, In Vivo Mouse Retinal Imaging System.

Novel gene therapy approaches for the eye are commonly tested initially in mouse or rodent models of disease.1–8 Most studies currently use some form of recombinant adeno-associated virus (AAV) or lentiviral vector to deliver genetic constructs to the retina or other regions of the eye. Delivery of wild-type genetic expression constructs is a first line approach to testing gene-based therapies for autosomal recessive conditions.3–6 More recently, posttranscriptional gene silencing agents as candidate therapeutics are being tested for treatment of autosomal dominant conditions in rodent models of disease.2,9 A popular delivery route for vectors for retinal gene therapy is by way of subretinal injection,6–8 which opens the subretinal space (separating outer segments of photoreceptors from the apical microvilli of the RPE) to permit efficient transduction of photoreceptors and/or RPE cells.10 Use of off-axis illumination common in standard clinical surgical microscopes poorly satisfy the entrance numerical aperture of the small mouse eye (diameter ∼3 mm) with a small dilated pupil aperture (≈1 mm), which makes it difficult to deliver robust input energy to the retinal surface to achieve quality specular imaging. Hence, vector delivery is often performed without direct or immediate visual feedback, and success and extent of delivery is often assessed on the basis of crude optical changes of the retinal red reflex, without clear delineation of the position of a subretinal fluid bleb, or post hoc through tissue expression analysis (e.g., enhanced green fluorescent protein [EGFP]). We considered and tried various commercial systems for mouse eye imaging. A critical factor in efficient imaging is the delivery of light through the pupil to illuminate the intraocular contents. The small size of the mouse eye and tiny dilated pupil constrain design of a microscope with a large working distance, to allow surgical manipulations, while viewing the retina by high-quality imaging. Prior, long-working distance systems used human surgical microscopes or microscopes with ring lights, which all use off-axis spatially broad sources that cannot be efficiently collected in order to deliver condensed light energy into the small mouse pupil (Supplementary Fig. S1). Imaging systems that contact the surface of the eye (e.g., Micron IV; Phoenix Research Labs, Pleasanton, CA, USA), limit approaches for real-time surgical intervention (e.g., subretinal injections). Here, we designed and tested a long working distance, stereo microscope–based, optical system that couples input light as a broad band or band-limited small spatial cone of illumination on-axis into the dilated mouse pupil. We call this device a retinal imaging system (RIS). We successfully used this system in both neonatal and adult mice in vivo to accomplish: (1) real time subretinal and intravitreal injections in the neonatal mouse eye, (2) imaging of normal and degenerative mouse retinas, (3) visualization of AAV-transduced regions of mouse retina, (4) far-red viewing of the mouse retina (to avoid light damage), and (5) true real-time intravenous fluorescein angiography in the live mouse. This inexpensive, long working distance device with constant illumination allows real-time surgical intervention and imaging of the retina or anterior segment of the eye, and rivals the imaging properties of commercially available contact imaging tools for the rodent eye. A preliminary version of these results were presented in abstract/poster format (Butler MC, et al. IOVS 2010;51:ARVO E-abstract 3103).