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. 2020 Oct 6;14:556700. doi: 10.3389/fnins.2020.556700

FIGURE 4.

FIGURE 4

Analysis of photoreceptors. (A) Exemplary immunofluorescence pictures of opsin (red) staining for L-cones and rhodopsin (green) staining for rods in photoreceptor outer segments. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) Fewer opsin+ cells were found in control compared to native retinas (p = 0.02). The filter and tweezers method showed, compared to native retinas, a better preservation over a cultivation period of 4 days. The number of opsin+ cells was significantly higher in tweezers samples (p = 0.04) compared to the control ones, while no changes were noted between filter and control retinas. A severe loss of opsin+ cells was discovered in the control compared to native samples after 8 days (p < 0.001). In the novel methods filter and tweezers, the number of opsin-labeled cells was comparable to native retinas. When comparing filter and tweezers samples (both: p < 0.001) to the controls, more opsin+ cells could be detected. (C) OPSIN expression was not altered at 4 days. OPSIN mRNA expression in tweezers samples was significantly upregulated compared to control retinas at 8 days (p = 0.002). (D) The rhodopsin signal found in control (p < 0.001) and filter samples (p = 0.01) was significantly less intense at 4 days compared to that in native samples. A significantly higher signal intensity was documented in tweezers (p < 0.001) and filter retinas (p = 0.047) compared to the controls. Moreover, a higher rhodopsin signal intensity was observed in tweezers retinas compared to filter samples (p = 0.04) at 4 days. At 8 days of cultivation, all three methods showed a significantly diminished rhodopsin intensity compared to native samples (all: p < 0.001). (E) RHODOPSIN mRNA expression was not altered at 4 days. Quantitative real-time PCR (RT-qPCR) examination of RHODOPSIN demonstrated an upregulation in tweezers samples compared to control retinas at 8 days (p = 0.02). OS, photoreceptor outer segments; ONL, outer nuclear layer. Scale bar: 20 μm, values are mean ± SEM for immunofluorescence (IF) and median ± quartile + min/max for RT-qPCR. IF: n = 9–10/group; RT-qPCR: n = 5/group. *p < 0.05 and ***p < 0.001 vs. native group; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. controls; ¥p < 0.05 vs. filter group.