Localized Photoreceptor Ablation Using Femtosecond Pulses Focused With Adaptive Optics

Abstract

Purpose The development of new approaches to human vision restoration could be greatly accelerated with the use of nonhuman primate models; however, there is a paucity of primate models of outer retina degeneration with good spatial localization. To limit ablation to the photoreceptors, we developed a new approach that uses a near-infrared ultrafast laser, focused using adaptive optics, to concentrate light in a small focal volume within the retina. Methods In the eyes of eight anesthetized macaques, 187 locations were exposed to laser powers from 50 to 210 mW. Laser exposure locations were monitored for up to 18 months using fluorescein angiography (FA), optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), adaptive optics scanning laser ophthalmoscope (AOSLO) reflectance imaging, two-photon excited fluorescence (TPEF) ophthalmoscopy, histology, and calcium responses of retinal ganglion cells. Results This method produced localized photoreceptor loss with minimal axial spread of damage to other retinal layers, verified by in-vivo structural imaging and histologic examination, although in some cases evidence of altered autofluorescence was found in the adjacent retinal pigment epithelium (RPE). Functional assessment using blood flow imaging of the retinal plexus and calcium imaging of the response of ganglion cells above the photoreceptor loss shows that inner retinal circuitry was preserved. Conclusions Although different from a genetic model of retinal degeneration, this model of localized photoreceptor loss may provide a useful testbed for vision restoration studies in nonhuman primates. Translational Relevance With this model, a variety of vision restoration methods can be tested in the non-human primate.

Imaging Transplanted Photoreceptors in Living Nonhuman Primates with Single-Cell Resolution

“…Following a preliminary attempt (see Note S3 ), we decided not to pursue this approach further for transplantation studies, although it was possible that we could have reduced these problems with a lower light exposure. Instead, we adopted a laser lesion method recently developed in our laboratory and described in detail in Dhakal et al (2020) . Selective photoreceptor ablation was observed after exposing host photoreceptors to ultrafast pulses of light delivered using the adaptive optics system.…”

“…While inherited models of retinal degeneration have recently been identified in nonhuman primates ( Moshiri et al., 2019 ; Peterson et al., 2019 ), at present there is no readily available genetic primate model of retinal degeneration. In this study, we used a recently developed model of selective photoreceptor ablation by using femtosecond pulses of near infrared (NIR) light onto the retina with adaptive optics ( Dhakal et al, 2020 ). This method of photoreceptor ablation leads to minimal disruption of the surrounding tissue, including the retinal pigment epithelium (RPE).…”

Optogenetic therapy restores retinal activity in primate for at least a year following photoreceptor ablation

“…We have previously demonstrated that even in areas of the primate retina where photoreceptor ablation was performed under overlying RGCs, their function was not impaired. 17 In contrast to much of the remodeling literature, in the present study the therapeutic ChrimsonR was present before photoreceptor ablation and was periodically stimulated for the purposes of this study. Although experimental optogenetic activation was for no more than 15 min during relatively infrequent imaging sessions, this could have been sufficient to preclude remodeling that might be driven in part by loss of visually driven activity in inner retina.…”

“…In this study, we use an acute photoreceptor ablation approach 17 to create a micro-scotoma in the adult primate, depriving a subset of foveal RGCs of their photoreceptor input. We use fluorescence fundus imaging and structural and functional AOSLO to follow optogenetic expression and responsivity of RGCs with and without photoreceptor input for 12 weeks after photoreceptor ablation and in a second animal demonstrate that optogenetic-mediated RGC activity persists for at least 12 months after the loss of photoreceptor input.…”

Developing Non-Human Primate Models of Inherited Retinal Diseases

“…Initial studies suggested that longer wavelength lasers result in damage of the outer retina layers, while shorter wavelength lasers damage all retinal layers [ 8 ]. Other studies have combined femtosecond laser with adaptive optics to achieve focal photoreceptor ablation in NHPs [ 9 ]. The ability to selectively limit damage to the outer retina layer can be useful as RPE disruption compromises the blood–retina barrier and evokes a strong immune response, whereas damage to the inner retina impedes functional re-circuiting.…”