In vivo imaging of functional photoreceptor recovery after injury and its relationship to the time courses of microglia and Müller cell activation

Abstract

Purpose :
To visualize the structural and functional remodeling of the mouse retina after local laser injury in vivo and assess the role of microglia and Müller cells in the recovery process.

Methods : Mice (c57Bl6/J, Cx3cr1^+/GFP, and Ai9-tdT;PDGFRa-CRE, Jackson Labs) were handled according to NIH and ARVO guidelines using a protocol approved by the UC Davis IACUC. Photoreceptor function, microglia, and Müller cells were imaged in vivo with custom-built combined scanning laser ophthalmoscope and optical coherence tomography system, and an adaptive optics SLO system. A near infrared laser (~8 mW, 860 nm, 2 minutes) caused localized photoreceptor damage as assessed by changes in OCT light scattering and histology. Immunohistochemistry was performed to support in vivo results.

Results : Near infrared (NIR) radiation caused photoreceptor damage that was immediately apparent as an increase in light scattering measured by OCT. Over days, the damaged area shrunk in size and by 3 weeks, only a small scar in the center of the damaged region remained. Using an established optophysiological protocol that measures rod phototransduction (Zhang et al. 2017 PNAS), we found that photoreceptors in the damaged locus failed to signal immediately after damage, but began to recover function after 14 days. At 3 weeks, the magnitude of the light response in the damaged region was 75% of the undamaged photoreceptor response. Microglia responded to the local damage by migrating both laterally and axially through the retina over hours, forming a dense cluster cells over the entire damaged locus that persisted for a week post-exposure. Müller cells initially vanished from the damaged area, but began to reappear 2 weeks later. At 3 weeks, highly GFAP+ Müller cells were clustered in the core of the damaged region. Only after microglia left and Müller cells re-populated the damaged area did functional photoreceptor recovery occur.

Conclusions : The NIR damage model reveals that a region of severe retinal damage can recover function. The functional recovery occurs after both the withdrawal of activated microglia and the establishment of the Müller cell response. The precise 3D spatiotemporal localization of the injury locus and the responses of microglia and Müller cells in this model provides a highly reliable foundation for studies investigating the local mechanisms of retinal repair.

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