Abstract
Purpose :
Microglia are the primary resident immune cells in the retina and may signal to systemic responders such as neutrophils in the early stages of inflammation. However, little is known about the interactions between these cell types in retinal damage. Here we monitor the progressive response of microglia and neutrophils from hours to days after photoreceptor (PR) laser injury using both in vivo imaging and post-mortem confirmation in mice.
Methods :
Outer retinal damage was induced by placing a 488 nm laser (1.12 mW) focused at the PR outer segments. Immune cells were tracked in 7 mice with adaptive optics scanning light ophthalmoscopy (AOSLO) and optical coherence tomography (OCT, Heidelberg Spectralis). Retinas were imaged at baseline, days 1, 3, 7 and 2 months post-damage. Another 7 mice were evaluated with post-mortem histology of whole-mount retinal tissue imaged with a Nikon A1 confocal microscope. Cx3CR1-GFP mice (Jax #5582) were used to track fluorescent microglia, neutrophils were labeled with Ly-6G-647 antibody and DAPI labeled nuclei.
Results :
OCT and confocal AOSLO showed brightening at the OPL/ONL interface within one hour of laser exposure, expanding deeper and becoming brighter over 24 hrs. Phase-contrast AOSLO revealed local disruption of PR somas that lasted out to 2 months (Fig. 1). Despite the damage, retinal capillaries remained perfused.
Fluorescence AOSLO and ex vivo histology showed laterally ramified microglia at baseline. 24 hours post insult cells showed axial protrusions into the ONL, becoming amoeboid by 3-7 days with somas migrating into the ONL and the distal segments of PRs. At 2 months, microglia were no longer aggregated and cells were distributed similar to baseline conditions. Despite the strong microglial response, neutrophils were not detected at the lesion site and those found in the retinal tissue were confined within the retinal vasculature (Fig. 2).
Conclusions :
In response to laser damage, microglia become regionally active, and yet, we saw no evidence for neutrophil recruitment at any time point from hours to months after injury. Despite confirmed loss of PR nuclei, and reactive microglia, this would suggest that the sterile nature of the laser damage does not invoke a neutrophil response. Elaborations on the model may provide new insights to the temporally complex cascade of inflammatory cell types that respond to injury.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.