In vivo microglia imaging during photoreceptor degeneration

Abstract

Purpose: In mice lacking visual arrestin (Arr1-/-), prolong phototransduction signaling in rods causes rapid photoreceptor degeneration and activation of retinal microglia (Levine et al., 2014). Within 12 hrs of light onset, microglia abandon their highly ramified "resting" state and migrate to the outer nuclear layer (ONL) where they subsequently phagocytose photoreceptor somata. This change in morphology and localization follows a consistent pattern, allowing discrete morphological staging and quantification over time to help determine the role of microglia during the progression of degeneration.

Methods: Arr1-/- were crossed with Cx3cr1^GFP/GFP mice, and Arr1-/- Cx3cr1^+/GFP mice used for in vivo imaging. Mice were born and raised in complete darkness before exposure to 200 lux continuous white light for fixed times ranging from 0 to 72 hrs. Scanning Laser Ophthalmoscopy with and without Adaptive Optics (AO-SLO) was used to quantify number and density and to determine the morphology of microglia across the retinal surface and within the different retinal layers during degeneration. Phase Variance Optical Coherence Tomography (pvOCT) was used to unequivocally co-register microglia radial position in respect to retinal capillary plexus. Microglial staging (S1 to S4) was defined by morphological criteria and laminar residence.

Results: AO-SLO fluorescence imaging revealed marked changes in cell morphology and localization after light onset. Within 12 hrs, microglia progressed from S1 stellate cells with small somata and fine dynamic processes to migratory S3 cells with elongated somata and polarized branching directed toward the ONL. S2 microglia have thicker, less branched processes, but were not observed in vivo at 12 hrs, likely due to the rapid progression from S1 to S3. S4 ameboid cells with large somata and little branching appeared by 24 hrs, coinciding with the presence of CD68+ cells found to be engulfing photoreceptor somata in fixed sections.

Conclusions: AO-SLO imaging of retinal microglia during photoreceptor degeneration can be used to quantitatively characterize microglial dynamics in vivo in the changing microenvironment during rapid photoreceptor degeneration. While AO-SLO studies nicely complement standard IHC analyses, they also provide the distinct advantage of repetitive imaging of individual microglia, and the assessment of directionality and rate of movement within a given animal.