Journal: Frontiers in Neuroscience

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

doi: 10.3389/fnins.2025.1596464

Figure Lengend Snippet: Validation of Rbpms2 as a pan-retinal ganglion cell (RGC) marker in the teleost. (A) UMAP projection of the 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 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 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.

Article Snippet: Next, murine and fish retinal WMs were immunohistochemically stained for RBPMS (rabbit anti-RBPMS, PhosphoSolutions, AB_249225, 1:250) or Rbpms2 (rabbit anti-Rbpms2, Abcam, Ab181098 , 1:200), respectively.

Techniques: Biomarker Discovery, Marker, Expressing, Transgenic Assay, Staining, Labeling

Journal: Frontiers in Neuroscience

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

doi: 10.3389/fnins.2025.1596464

Figure Lengend Snippet: 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.

Article Snippet: Next, murine and fish retinal WMs were immunohistochemically stained for RBPMS (rabbit anti-RBPMS, PhosphoSolutions, AB_249225, 1:250) or Rbpms2 (rabbit anti-Rbpms2, Abcam, Ab181098 , 1:200), respectively.

Techniques: Labeling

Journal: Frontiers in Neuroscience

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

doi: 10.3389/fnins.2025.1596464

Figure Lengend Snippet: 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.

Article Snippet: Next, murine and fish retinal WMs were immunohistochemically stained for RBPMS (rabbit anti-RBPMS, PhosphoSolutions, AB_249225, 1:250) or Rbpms2 (rabbit anti-Rbpms2, Abcam, Ab181098 , 1:200), respectively.

Techniques: Staining, Control, MANN-WHITNEY

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: Intravitreal injection of SCGF-β protects RGCs after optic nerve crush. A Human SCGF-β or PBS was intravitreally injected into the adult mouse eye right after optic nerve crush. The retina was explanted and immunostained 5 days post cush. B RBPMS + RGCs under SCGF-β administration showed significantly higher survival, compared with the PBS treatment. * P < 0.05. ONC, optic nerve crush. Error bar denotes SD

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Injection

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: RGCs enhance iPSC’s SCGF-β release via IL-12(p70). A - B IL-12(p70) was detected in RGC supernatant, not in RGC medium, through multiplexed antibody-based assays and confirmed by ELISA. C - D Compared to the control (“No IL-12(p70)” group), significant upregulation of SCGF-β (via ELISA ) and CLEC11A mRNA (via qRT-PCR ) were observed in iPSCs treated with 2.5, 5, and 10 ng/mL of IL-12(p70). The expression peaked in the 5 ng/mL IL-12(p70)-treated group. E RGCs were cultured in the RGC medium with different dosages of IL-12(p70) administration. A significantly greater cell viability was not found until treated with 40 ng/mL of IL-12(p70). * P < 0.05. Error bar denotes SD. NS, nonsignificant

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: iPSC-derived SCGF-β promotes RGC survival via upregulation of ngn2. A - B RT-qPCR results showed a significant increase of ngn2 mRNA in RGCs cocultured with iPSCs or treated with SCGF-β. C - D Overexpression of ngn2 in RGCs cultured in the RGC medium for 1 week significantly increased RGC viability. E Knockdown of ngn2 in RGCs cultured for 1 week does not significantly alter RGC survival, whether treated with SCGF-β or not. * P < 0.05. Error bar denotes SD

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Derivative Assay, Quantitative RT-PCR, Over Expression, Cell Culture, Knockdown

Journal: Stem Cell Research & Therapy

Article Title: Retinal ganglion cells induce stem cell-derived neuroprotection via IL-12 to SCGF-β crosstalk

doi: 10.1186/s13287-025-04198-5

Figure Lengend Snippet: Overexpression of ngn2 protects endogenous and transplanted RGCs in vivo. A AAV2-ngn2-EGFP or AAV2-EGFP particles were intravitreally injected 2 weeks into adult mouse eyes 2 weeks before optic nerve crush. Retina explants were harvested and immunostained 2 weeks after the optic nerve crush. Surviving RGCs were labeled with RBPMS (red). B A significantly greater RBPMS + RGC number was found on retina explants from the AAV-ngn2 treated group. C The EGFP and mCherry were used to label the entire transplanted and ngn2-overexpressing donor mouse RGCs, respectively. Ngn2-mCherry-overexpressing mouse RGCs that were transplanted into adult rat eyes shows significantly higher survival rates, compared with the negative control RGC transplant 1 week after transplantation in vivo. D & E Difference between transplanted RGC survival rates and average neurite lengths with and without ngn2 overespression was quantified . Donor RGCs overexpressing ngn2-mCherry grew significantly longer neurites than those from negative control-RGC transplantation in vivo. * P < 0.05, paired t-test. ONC, optic nerve crush; NC, negative control; OE, overexpression. Error bar denotes SD

Article Snippet: The flat mounted samples were permeabilized with 0.3% Triton X-100 (Cat. #T9284; Sigma-Aldrich) for 20 min, blocked with 5% normal goat serum (Cat. #16,210,064; Invitrogen, San Diego, CA, USA) in PBS for 1 h. For the retinas from optic nerve crush, the samples were incubated with a rabbit polyclonal anti-RNA Binding Protein, mRNA Processing Factor (RBPMS) antibody (1:500; Cat. #1832-RBPMS; PhosphoSolutions, Aurora, CO, USA) overnight at 4 °C, rinsed three times with PBS, and then incubated with Alexa Fluor 555-tagged secondary antibody (1:500; Cat. #A-21428; Life Technologies) overnight, again.

Techniques: Over Expression, In Vivo, Injection, Labeling, Negative Control, Transplantation Assay