Cellular-scale evaluation of induced photoreceptor degeneration in the living primate eye

Abstract

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinicallyrelevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.

Optogenetic restoration of retinal ganglion cell activity in the living primate

“…The eye was enucleated and the tissue postfixed and dehydrated before plastic embedding and sectioning into 2.5 μm sections. Full details of the protocol can be found in Walters et al 23 . A two part hematoxylin and eosin stain was performed to label nuclei blue and cytoplasm pink, allowing assessment of structural damage.…”

Localized Photoreceptor Ablation Using Femtosecond Pulses Focused With Adaptive Optics

“…TPEF imaging was conducted after the laser exposure for functional measurement of the visual cycle in photoreceptors as described elsewhere. 24 , 25 Briefly, a Ti:Sapphire femtosecond laser (55 fs, 80 MHz, 730 nm, Mai Tai XF-1 with DeepSee attachment; Spectra-Physics, Santa Clara, CA, USA), compensated for second-order dispersion arising from both the optics of the AOSLO and the eye, was used to excite TPEF; the maximum power at the cornea was 3.5 to 4.5 mW. An 840-nm superluminescent diode was used for wavefront sensing with a Shack-Hartmann wavefront sensor, and a maximum power of 45 µW was incident on the cornea.…”

“…Additionally, the 730-nm reflectance channel was used for nonconfocal multioffset imaging, which has been described elsewhere. 25 A third PMT for TPEF imaging (excitation: 730 nm; detection: 400–550 nm) was used. The TPEF time course data was adapted using a method described elsewhere 26 with an exponential function y = Δ F / F e − t / t + 1, where τ is the time constant of TPEF increase and Δ F / F is the relative increase in TPEF.…”

Imaging Retinal Activity in the Living Eye

“…Imaging the visual cycle may be useful in quantifying photoreceptor function, as demonstrated in three macaque models of altered outer retinal function. In a macaque model of induced retinal degeneration, photoreceptors missing their outer segments showed little change in TPEF upon stimulation (Walters et al 2019) Similarly, phototoxic damage to select cones may be identified by a cell-specific decrease in TPEF intensity and altered TPEF time course after visual stimulation (Schwarz et al 2018). The rate at which TPEF intensity changes with visual stimulation is also slowed in the cones of macaques breathing 10% versus 100% O 2 .…”