Figure 1. Mitochondrial fission and loss of cristae following exposure to elevated hydrostatic pressure. Differentiated RGC-5 cells were exposed to elevated hydrostatic pressure (30 mmHg) for 3 days and stained with MitoTracker Red. Non-pressurized control cells show a typical filamentous and fused mitochondrial network (A). Pressurized cells show mitochondrial fission, which is characterized by the conversion of tubular fused mitochondria into isolated small organelles (B). Electron micrographs of thin sections of RGC-5 cells show normal, elongated forms of mitochondria in non-pressurized control cells (C), whereas elevated hydrostatic pressure produces smaller mitochondria (arrow) and mitochondria with abnormal, severe cristae depletion (arrowheads; D). Closely apposed smaller mitochondria were also observed, such as the six shown in panel E, suggesting that fission had occurred. To quantify the observation of severe cristae depletion in the mitochondria exposed to elevated hydrostatic pressure, the cristae density in tomographic volumes was determined after segmentation. This parameter was calculated by dividing the sum of the cristae volumes by the mitochondrial volume for each mitochondrion (F). There were 13 control (from two experiments) and 14 pressurized mitochondria (from two experiments) fully segmented into constituent compartments and analyzed (The asterisk indicates significance at p<0.001 compared to non-pressurized cells). Data represent the means±SD. Size bar represents 20 µm (A and B) and 500 nm (C-E).