Abstract
Purpose:
Apply ultrahigh resolution spectral domain optical coherence tomography (UHR-SD-OCT) to assess both the in vivo progress of outer retinal photoreceptor degeneration in mouse models, and to exploit the instrument to capacity to aid in delivery of gene therapy vectors into the subretinal space.
Methods:
We developed a parafocal, paraxial Greenough-type stereo microscope as an optimized non-contact retinal imaging system (RIS) for real time subretinal surgery in the anesthetized mouse. The RIS is optimized for the small mouse pupil, operates in various modes (bright field, fluorescence, infrared) with color CCD camera image capture, and has long working distance for surgical manipulations. The UHR-SD-OCT (Bioptigen Inc) operates with near infrared light (~850 nm), has optics optimized for the mouse eye, and resolution of 2x2x2 micron voxels. Different mouse retinal degeneration models and controls were used. Subretinal injections of AAV vector or nanoparticles (NP) are accomplished with pulled glass microneedles advanced with micromanipulator by trans-scleral and trans-choroidal approach and positioned beneath the retina before a calibrated pressure injector delivers into the subretinal space.
Results:
The RIS presents a narrow beam through the dilated pupil to illuminate the retina, and the optic nerve, vasculature, and posterior pole are crisply visualized, while the entire anterior retina can be observed through eye positioning. Normal and degenerative retinal phenotypes are followed over time by microscopic direct fundoscopic imaging. The RIS and micromanipulator-based delivery system allows real-time visualization and tight control over subretinal injections in neonatal (~P14) or adult mice. The UHR-SD-OCT is used to immediately determine success of a subretinal injection. Gold nanorods, useful for infrared OCT imaging, mark regions expected to be transduced by viral vector. UHR-SD-OCT permits visualization of all retinal layers, and allows rigorous statistical quantitation of outer nuclear layer (ONL) thickness as measure of photoreceptor vitality to assess degeneration or rescue.
Conclusions:
Advanced imaging devices (RIS, UHR SD-OCT) are powerful tools to assist real time subretinal surgery in mice eyes, and to assess the state of subretinal injection, and vitality of photoreceptors over time in animal models of retinal degeneration or in preclinical gene therapy trials.
Keywords: 538 gene transfer/gene therapy •
607 nanotechnology •
551 imaging/image analysis: non-clinical