Investigation of the two types of cellular connections of Schlemm's canal inner wall cells and their role in giant vacuole and pore formation by serial block-face scanning electron microscopy
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PURPOSE: To determine, under flow conditions, whether reduced connections between Schlemm’s canal (SC) inner wall (IW) and juxtacanalicular tissue (JCT) cells play a role in giant vacuole (GV) formation; and whether decreased amount of cell margin overlap between adjacent IW cells promotes paracellular pore formation using serial block-face scanning electron microscopy (SBF-SEM). METHODS: Normal human eyes were immersion-fixed (0 mmHg, N=2) or perfusion-fixed (15 mmHg, N=1). Frontal and radial sections of SC were processed for SBF-SEM. IW and JCT cells, GVs, and pores were 3D-reconstructed. In each IW cell, total number of connections with underlying JCT cells/matrix was determined. Total cell margin length (TCML) and zero-overlap length (ZL) of each IW cell were measured to calculate percent zero-overlap length (PZL=ZL/TCML). All data were compared between the eyes fixed at 0 and 15 mmHg. RESULTS: Total number of IW/JCT connections in individual IWs significantly decreased in the eye fixed at 15 mmHg (33±5, N=5 cells) compared to those fixed at 0 mmHg (189±12, N=4 cells, p<0.001). The summed GV volume in individual cells significantly increased in the eye fixed at 15mmHg (218.03±19.65 μm3) compared to those fixed at 0 mmHg (82.33±27.22 μm3, p=0.0043). PZL increased 26.68% (p=0.001) in the eye fixed at 15mmHg vs. those fixed at 0mmHg, and all paracellular pores were found only in regions where the overlap length was 0 μm. CONCLUSIONS: Cellular connections between IW/JCT and IW/IW cells play a role in GV and pore formation in normal human eyes under flow conditions. Our results provide a baseline for future comparison with primary open angle glaucoma eyes.