Dynamic changes in cell size and corresponding cell fate after optic nerve injury

Abstract

Identifying disease-specific patterns of retinal cell loss in pathological conditions has been highlighted by the emergence of techniques such as Detection of Apoptotic Retinal Cells and Adaptive Optics confocal Scanning Laser Ophthalmoscopy which have enabled single-cell visualisation in vivo. Cell size has previously been used to stratify Retinal Ganglion Cell (RGC) populations in histological samples of optic neuropathies, and early work in this field suggested that larger RGCs are more susceptible to early loss than smaller RGCs. More recently, however, it has been proposed that RGC soma and axon size may be dynamic and change in response to injury. To address this unresolved controversy, we applied recent advances in maximising information extraction from RGC populations in retinal whole mounts to evaluate the changes in RGC size distribution over time, using three well-established rodent models of optic nerve injury. In contrast to previous studies based on sampling approaches, we examined the whole Brn3a-positive RGC population at multiple time points over the natural history of these models. The morphology of over 4 million RGCs was thus assessed to glean novel insights from this dataset. RGC subpopulations were found to both increase and decrease in size over time, supporting the notion that RGC cell size is dynamic in response to injury. However, this study presents compelling evidence that smaller RGCs are lost more rapidly than larger RGCs despite the dynamism. Finally, using a bootstrap approach, the data strongly suggests that disease-associated changes in RGC spatial distribution and morphology could have potential as novel diagnostic indicators.

Quantification and image-derived phenotyping of retinal ganglion cell nuclei in theneemouse model of congenital glaucoma

“…Urcola et al described this as "hypertrophy". Because RGC somal and nuclear areas have previously been shown to be positively correlated (Davis et al, 2020;Janssen et al, 2013), it seems likely that the "hypertrophy" observed by Urcola et al, and "spreading" described here, are the same phenomenon. Davis et al (Davis et al, 2020) observed RGC nuclear enlargement in the early stages following partial optic nerve transection and hypothesized that it might involve metabolic responses of the injured RGCs.…”

“…Because RGC somal and nuclear areas have previously been shown to be positively correlated (Davis et al, 2020;Janssen et al, 2013), it seems likely that the "hypertrophy" observed by Urcola et al, and "spreading" described here, are the same phenomenon. Davis et al (Davis et al, 2020) observed RGC nuclear enlargement in the early stages following partial optic nerve transection and hypothesized that it might involve metabolic responses of the injured RGCs. Because we have observed spreading in not only glaucomatous RGCs, but also in non-RGCs of mice with glaucoma and in RGCs of healthy WT mice, we propose a more passive response-though future studies to study this phenomenon are needed.…”

“…RGCs are significantly larger in the peripheral retina, an area of relatively low cell density compared to the central retina, where RGCs are smaller and more numerous (Drager and Olsen, 1981;Urcola et al, 2006). RGC enlargement in some stages of disease has been previously observed in multiple studies (Davis et al, 2020;Hedberg-Buenz et al, 2016a;Moore and Thanos, 1996;Urcola et al, 2006). For example, among surviving RGCs in a rat model of chronic IOP elevation induced by microbead injection (Urcola et al, 2006), in which a loss of 27.2% of RGCs after 24 weeks led to a 11.9% increase in RGC somal area-a subtle change in size in response to modest changes in RGC density similar to those observed here.…”

“…Accordingly, the data most specifically relate to the surviving RGC population that still expresses BRN3A. Although Brn3a appears to be transcribed in all sub-classes of RGCs (Rheaume et al, 2018), most studies find that immunolabeling fails to identify some (~3-15%) RGCs (Davis et al, 2020;Galindo-Romero et al, 2011;Rodriguez et al, 2014). Thus, RGCs that either lost, or never had, detectable BRN3A were potentially confounding.…”

“…The results point to competing events influencing nuclear size, with spreading of multiple cell types occurring with modest decreases in RGC density and a preferential loss of large RGC nuclei in moderate to severe cases of RGC loss due to glaucoma. Given the progress in refining approaches using automated image analysis to quantify RGC soma (Danias et al, 2006;Dordea et al, 2016;Guymer et al, 2020;Hedberg-Buenz et al, 2016a;Hedberg-Buenz et al, 2016b;Masin et al, 2021;Salinas-Navarro et al, 2009) and axons (Bosco et al, 2015;Mysona et al, 2020;Reynaud et al, 2012;Ritch et al, 2020;Teixeira et al, 2014;Zarei et al, 2016) it appears that the approach that we, and others (Davis et al, 2020), have begun to utilize to phenotype RGCs in these automated ways may make additional discoveries. Although studies to date have analyzed only a small number of parameters that were all related to nuclear size, future experiments could likewise analyze many additional features, some of which may also be beyond the practical limit for humans to have been led to suspect as important based only on visual observation.…”