Spatially localized photoreceptor ablation in macaque using a femtosecond laser and adaptive optics

Abstract

Purpose : The development of vision restoration therapies could be accelerated by using non-human primates because of their similarity to humans. Unfortunately, there is a paucity of primate models with selective damage to photoreceptors. Chemical and photothermal strategies to damage photoreceptors have poor spatial localization, typically damaging other retinal layers. We developed a new approach that provides superior control of the spatial extent of the ablated retina.

Methods : We combined two methods to improve spatial localization: the high resolution provided by adaptive optics and the nonlinearity of light-tissue interactions provided by ultrafast laser. In 3 eyes of 2 anesthetized male macaques, an adaptive optics scanning light ophthalmoscope (AOSLO) focused a femtosecond laser (730 nm, 55 fs, 80 MHz, ~3.5 µm spot size, 94 ms exposure duration) on the outer nuclear layer. 45 locations were exposed at a 27 Hz frame rate, each with an area of 0.7°x0.8°. Power was varied from 50-210 mW. Fluorescein angiography was performed within hours after exposure and at 24 and 72 hours. Exposed locations were monitored over 6 months using OCT, SLO, and AOSLO reflectance and two-photon excited fluorescence (TPEF) imaging. Histology was performed in one eye. In an additional female macaque, AOSLO calcium imaging assessed the function of retinal ganglion cells directly above photoreceptor lesions.

Results : Average powers below 68 mW did not produce visible damage in any in vivo imaging modality; 80 to 120 mW caused partial loss of photoreceptors, and above 130 mW completely ablated all photoreceptors. Consistent with histological results, four lines of in vivo evidence suggest the damage was laterally and axially confined to the photoreceptor layer: early fluorescein leakage was not seen at 72 hours, a sharp border was seen between ablated and waveguiding cones, retinal capillaries vitread of the lesion appeared normal, and light evoked calcium responses in ganglion cells above the lesions were unaffected.

Conclusions : The use of an ultrafast laser in conjunction with adaptive optics produced highly localized, selective ablation of the photoreceptor layer. Though different from a genetic model of retinal degeneration, our approach can be readily deployed in vision restoration techniques such as photoreceptor precursor transplantation and optogenetics that require an intact RPE and inner retina.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.