Figure 7
RGC survival test and visualization of apoptotic cells by fluorescence microscopy. (A–E) RGC survival using RGC marker (NeuN) in whole mount retinas. (A) Confocal images of NeuN-labeled RGCs in a control eye incubated at 10 mmHg. (B) Density of NeuN-positive RGCs was reduced at 75 mmHg compared to 10 mmHg. (C,D) Administration of AlloP (0.1 μM) alone (C) or 24SH (0.1 μM) alone (D) partially protected RGCs from pressure-induced injury. (E) Combination of AlloP (0.1 μM) and 24SH (0.1 μM) enhanced RGC survival under hyperbaric conditions. (A–E) are at the same magnification. Scale bars, 200 μm. (F) The graph presents the number of NeuN-positive cells in the whole mount retina under each experimental condition. Statistical differences were analyzed using Dunnet’s or Tukey’s multiple comparison test (*p < 0.05). (G–K) Merge of differential interference contrast images and fluorescence images using DAPI and TUNEL-fluorescent staining of retinas. (G) At 10 mmHg, few TUNEL-positive cells (arrows) can be observed within the ONL. (H) At 75 mmHg, there was a marked increase of TUNEL-positive cells in the GCL as well as some cells in the INL and ONL. (I,J) Administration of 0.1 μM AlloP alone (I) or 0.1 μM 24SH alone (J) significantly decreased a number of TUNEL-positive cells at 75 mmHg. (K) Combination of 0.1 μM AlloP and 0.1 μM 24SH almost completely inhibited apoptosis at 75 mmHg. A few TUNEL-positive cells (arrows) can be observed within the INL and ONL. a-e are at the same magnification. Scale bars, 30 μm. (L) The graph presents the number of TUNEL-positive RGCs per 200 μm of the retinal section. Statistical differences were analyzed using Dunnet’s or Tukey’s multiple comparison test (*p < 0.05).
