Interestingly, reduced TM cell viability was seen to be highly heterogeneous (segmental) around the TM circumference, as quantified by the fraction of viable regions around the TM (28% after H
2O
2 exposure vs. 59% in control, untreated eyes) and the coefficient of variation of viable cell staining around the TM circumference. It is important to note that even control eyes showed appreciable segmental variation, an effect that was amplified by H
2O
2. We speculate that damage is more severe in regions of high flow during the 1-hour H
2O
2 exposure in organ culture. These findings were consistent with observations from histological analysis, in which both control and H
2O
2-treated TMs were found to have regions of reduced cellularity. In fact, masked histological scoring could not detect a consistent difference in cellularity between H
2O
2-treated and control tissue, likely due to the significant undersampling associated with studying sagittal sections at discrete circumferential locations. Further experiments are needed to understand this aspect of the response to H
2O
2. For example, studies incorporating a fluorescent tracker to track high outflow regions along with H
2O
2 treatment could determine whether there is a correlation between high flow and reduced viability.
41 Second, a more rigorous serial sectioning process of the entire TM would be necessary to fully track reduced cellularity following H
2O
2 treatment. Third, immunohistochemical analysis could be used to track key proteins associated with the TM to better identify and track changes occurring in the tissue as a result of the H
2O
2 treatment. These findings also highlight a general limitation of the organ culture approach—namely, the segmental loss of cells in control eyes.
56 Even though eyes were obtained fresh and were dissected by an experienced investigator, such segmental variation appears unavoidable and future studies should consider this effect.