Differential retinal ganglion cell resilience to optic nerve injury across vertebrate species

Abstract

Optic neuropathies comprise a diverse group of disorders that ultimately lead to retinal ganglion cell (RGC) degeneration. Despite varying etiologies, these conditions share a conserved pathological progression: axonal damage in the optic nerve triggers progressive RGC degeneration. Understanding species-specific differences in neuronal resilience is critical for identifying key survival mechanisms and potential neuroprotective targets. In this study, we compare RGC densities and survival rates following optic nerve crush (ONC) in three vertebrate models-mice, zebrafish, and killifish-under standardized experimental conditions. Transcriptomic analysis confirmed that, similar to RBPMS in mice, Rbpms2 serves as a pan-RGC marker in zebrafish and killifish. Using these markers, we reveal significant species-specific differences in RGC density, with fish species exhibiting over a 5-fold higher density than mice at equivalent life stage. Killifish also show an age-dependent decline in RGC density. Furthermore, we identify distinct injury responses across species: mice undergo rapid degeneration, losing ∼80% of their RGCs by day 14 after ONC; zebrafish maintain full RGC retention for 2 weeks before experiencing a loss of ∼12%; and killifish display a biphasic response to ONC, with young adults retaining two-thirds of their RGCs by day 21, while older fish exhibit a more pronounced second wave of RGC loss, ultimately preserving just over half of their RGCs by 21 days after injury. These findings highlight fundamental differences in neuroprotective capacity among species, providing a comparative framework to uncover molecular mechanisms governing RGC survival and to identify therapeutic strategies for treating optic neuropathies and neurodegeneration across diverse pathologies.

Keywords: killifish; mouse; neuroprotection; optic nerve crush; retina; retinal ganglion cell; survival; zebrafish.

FIGURE 1

Validation of Rbpms2 as a pan-retinal ganglion cell (RGC) marker in the teleost. (A) UMAP projection of the Li et al. (2024) dataset, showing the clusters of major retinal cell types in the adult mouse retina. (B) Rbpms is a pan-RGC marker showing homogenous expression across all RGCs in the murine retina (zoom on the RGC cluster on the right). (C) Contrary to Rbpms, Pou4f1, also known as Brn3a, does not show homogenous expression across all RGCs in mice. (D) tSNE projection of the Hoang et al. (2020) dataset (displaying only non-injured cells) revealing the major retinal cell types in the adult zebrafish retina. (E) rbpms2 is a pan-RGC marker showing homogenous expression across all RGCs in the adult zebrafish retina (zoom on the RGC cluster on the right). (F) isl2b, the most commonly used promoter in transgenic reporter lines for RGCs in zebrafish, does not show homogenous expression across all RGCs in adult zebrafish. (G) UMAP projection of the Bergmans et al. (2024) dataset, showing the clusters of major retinal cell types in the adult killifish retina. (H) As in zebrafish, rbpms2 emerges as the most homogenous marker for RGCs in the adult killifish retina (zoom on the RGC cluster on the right). (I) isl2b, like in zebrafish, does not show homogenous expression across all RGCs in adult killifish. (J) Representative micrographs of zebrafish retinal WMs, in which the RGCs are retrogradely traced with biocytin and immunostained for Rbpms2. Contrary to biocytin, Rbpms2 staining results in more homogenous, somatic labeling and labeled cells are not occluded by axonal bundles in the nerve fiber layer (arrows). Scale bar 25 μm. (K) Representative micrographs of killifish retinal WMs, in which the RGCs are retrogradely traced with biocytin and immunostained for Rbpms2. As in zebrafish, Rbpms2 staining results in homogeneous somatic labeling, while biocytin tracing is more heterogenous and RGCs are occasionally occluded by axon bundles (arrows). Scale bar 25 μm. AC, amacrine cell; BC, bipolar cell; HC, horizontal cell; MG, Müller glia; RBC, red blood cell; RGC, retinal ganglion cell; RPE, retinal pigment epithelium; V/E, vascular/endothelial; WMs, whole-mounts.

FIGURE 2

Different retinal ganglion cell (RGC) density in retinas of adult mice, zebrafish and killifish. (A–D) Scaled representation of retinas from young adult mice [10 weeks-old, (A)], young adult zebrafish [21 weeks-old, (B)], young adult killifish [6 weeks-old, (C)] and old killifish [18 week-old, (D)]. Mice have larger retinas than zebrafish and young killifish, while the retina of aged killifish is considerably larger than that of their younger counterparts. Scale bar 1 mm. (E–H) Representative micrographs of RGCs labeled with RBPMS [mouse, (E)] or Rbpms2 (fish), sampled from the temporal retina. Young adult zebrafish (F) and killifish (G) show a comparable density, higher than the one of old killifish (H) and young adult mice. Moreover, RGCs from the fish species are considerably smaller than the ones of mice. Scale bar 25 μm. (I) Automated quantification of the area of retinal WMs, revealing that unlike young adult fish, old killifish approach the size of murine retinas. (J) Automated quantification of RGC numbers in retinal WMs. Mice exhibit the lowest RGC count, with approximately 45,000 cells. In contrast, young fish possess around 70,000 RGCs. Aged killifish have the highest count, reaching approximately 125,000, nearly twice as many as young killifish. (K) Automated quantification of RGC density in retinal WMs. RGC density is considerably lower in mice compared to fish species. Notably, old killifish exhibit a significantly reduced RGC density compared to young adult fish. D, dorsal; N, nasal; RGCs, retinal ganglion cells; T, temporal; V, ventral; WMs, whole-mount.

FIGURE 3

Differential retinal ganglion cell (RGC) susceptibility to optic nerve injury in canonical vertebrate models. (A) Experimental timeline for the RGC survival experiment in young adult mice and zebrafish. RGC survival was evaluated at 7 and 14 days post-ONC in mice and at 7, 14, and 21 dpi in zebrafish. With assets from BioRender.com. (B) Representative micrographs of RPBMS-stained murine retinal WMs following ONC injury show substantial RGC loss at 7 dpi, which becomes even more pronounced by 14 dpi. Scale bar 25 μm. (C) Representative micrographs of Rpbms2-stained zebrafish retinal WMs following ONC injury. ONC injury leads to no appreciable loss of RGCs at 7 and 14 dpi, but a minor loss can be observed at 21 dpi. Scale bar 25 μm. (D) Quantification of RGC survival in adult murine WMs. ONC leads to the loss of over 50% of RGCs at 7 dpi, and a further one until 14 dpi, when only about 20% of the RGCs remain. (E) Quantification of RGC survival in adult zebrafish WMs. There is no significant loss of RGCs within the first 2 weeks after ONC. A small but significant decrease is measured at 21 dpi, with approximately 10% of the RGCs lost. Data from two independent experiments, presented as percentages relative to the median of uninjured retinas and presented as median ± 25–75th CI. Kruskal-Wallis ANOVA. P-values reported within the figure. CI, confidence interval; dpi, days post injury; ONC, optic nerve crush; RGCs, retinal ganglion cells; WMs, whole-mounts.

FIGURE 4

Biphasic retinal ganglion cell (RGC) loss in killifish after optic nerve crush injury. (A) Representative image of a young adult (6 weeks-old) and old killifish (18 weeks-old) and experimental timeline for the RGC survival experiment, where RGC survival is evaluated at 4, 7, 14, and 21 days following ONC injury. Scale bar: 1 mm. (B) Representative micrographs of Rbpms2-stained WMs of young adult (6 weeks-old) and old killifish (18 weeks-old). For both ages, an appreciable loss of RGCs is evident at 7 dpi with further loss at 21 dpi, when compared to uninjured age-matched control fish. (C) Quantification of RGC survival in adult killifish WMs shows a first wave of RGC loss at 4 dpi, with 20% of the RGCs lost in both age groups, and this loss remains steady through 7 dpi. A second wave of loss is observed at 14 dpi, with older fish losing more RGCs (50%) compared to young fish (40%). No further loss is detected at 21 dpi in either age group. (D) Quantification of RGC survival per retinal quadrant in adult killifish WMs reveals no significant differences in inter-quadrant RGC loss after ONC by 21 dpi for both young adult (6 weeks-old) and old (18 weeks-old) fish. Data from two independent experiments, presented as percentages relative to the median of their uninjured age-matched control and presented as median ± 25–75th CI. Two-way Kruskal-Wallis ANOVA with pairwise Mann-Whitney U tests (C), One-way Kruskal-Wallis ANOVA and post hoc Mann-Whitney U test with Bonferroni correction [(D), 06 weeks], One-way Welch ANOVA and post hoc Games-Howell test [(D), 18 weeks]. p-values reported within the figure for significant differences. CI, confidence interval; DN, dorsonasal; dpi, days post injury; DT, dorsotemporal; ONC, optic nerve crush; RGCs, retinal ganglion cells; VN, ventronasal; VT, ventrotemporal; WMs, whole-mounts.