Retinal ganglion cell resilience to injury indicated by cytoplasmic [Ca²⁺] levels

Abstract

Purpose : Retinal ganglion cells (RGCs) are the sole projection neurons from the retina to the brain, and injury to their axons with an optic nerve crush (ONC) causes significant RGC death over multiple weeks. However, a subset of RGCs survive the injury, and intrinsic characteristics promoting survival remain unclear. Manipulating RGC activity has been shown to increase regeneration after ONC, and as cytoplasmic [Ca²⁺] levels are influenced by activity, we hypothesized that ground state [Ca²⁺] levels could predict the survival of RGCs to ONC injury.

Methods : We used two-photon (2p) microscopy to visualize RGCs in-vivo at single-cellular resolution. To label all RGCs, we used an intersectional approach with VGlut2-Cre mice and intravitreal injections of an adeno-associated viral vector (AAV) expressing a Cre-dependent cytoplasmic [Ca²⁺] biosensor, Twitch-2b. We visualized individual RGCs and quantified their somatic resting [Ca²⁺] levels in the alive mouse and then performed ONC to assess RGC survival. We also tested two subtypes of RGCs with this paradigm, alpha RGCs (aRGCs; KCNG4-Cre) and intrinsically photosensitive RGCs (ipRGCs; OPN4-Cre).

Results : Baseline [Ca²⁺] levels varied greatly between RGCs within a single retina (Fig 1). We found that RGCs with high ground state [Ca²⁺] levels prior to ONC survived 3x better at two weeks post ONC (38 ± 1.2% survival, n=9 retinas, cells(c)=124) than those with low resting [Ca²⁺] (16 ± .1%, c=136) (p=.018). Cells that survived had significantly higher ground state [Ca²⁺] levels than those that died (p=4.6E-6). Two subtypes, aRGC and ipRGCs, had much higher ground state [Ca²⁺] levels compared to all RGCs (p=7.6E-13, p=1.7E-29). Within each subtype, cells with high baseline [Ca²⁺] had increased survival (aRGC: 63 ± 2%, n=12, c=132; ipRGC: 60 ± 2%, n=9, c=296) compared to those with low [Ca²⁺] (35 ± 4%, c=63; 47 ± 4%, c=49). Interestingly, for both subtypes, surviving cells had higher baseline [Ca²⁺] levels than those that died (p=.15, p=1E-4).

Conclusions : Our results for all RGCs and for each subtype, aRGCs and ipRGCs, are consistent with our hypothesis. Thus, we can conclude that high resting cytoplasmic [Ca²⁺] levels in the RGC soma indicate resilience to axon injury. Future directions include identifying a low resting [Ca²⁺] level RGC cohort and testing their survival after ONC.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Figure 1: In-vivo 2p image of RGCs expressing Twitch-2b. [Ca²⁺] is determined from YFP/CFP ratios.