Finally, we think that the functional assay is more PI-103 sensitive and robust in assessing the overall health of the light sensing portion of the neuroretina because functional assays evaluate the health status of the entire photoreceptor cells and their connections to other cell types not just the presence/absence of photoreceptor cell bodies in the ONL. The process of apoptosis consists of three phases: initiation, commitment, and degradation. The estimated duration from initiation to degradation ranges from 6 to 24 hours with the greatest variation in the initiation phase. The TUNEL assay detects cells that are at the degradation phase which is estimated to be about 5 hours. The clearance of TUNEL positive profiles in the developing rat cerebral cortex is about 3 hours. Because apoptosis is a conserved cell death process, we think that these parameters will apply to the rod cells in the P23H-1 rats. Monitoring apoptosis by in situ TUNEL assay provides a temporal snapshot of the health status of cells in the tissue. Understandably, cells that have not begun the degradation phase and those that have completed the degradation phase will be missed using this LY2835219 method and likely causes an undercount of the number of apoptotic cells. Nonetheless, the cell death index by TUNEL assay is useful to assess the therapeutic activities or pharmacodynamics of therapeutic agents because TUNEL+ profiles detected at each time point greater than 5 hours apart would be from cells that have not been counted previously. We demonstrated that CeNPs were effective in reducing apoptotic photoreceptor cells up to 21 days after intravitreal injection. The anti-apoptotic effect was most striking up to 7 days after CeNPs application when we observed 56% reduction of TUNEL+ profiles in the ONL. The data represent the numbers of TUNEL+ profiles in single 5 ��m thick retinal sections through the central part of the eye. If we extrapolate that number to the whole retina, the number of dying cells will be in the thousands every day. For example, the estimated TUNEL+ profiles in the whole retina at P22 would be ~62,000 at the time of tissue harvesting. If we sample the retina every six hours, we would observe a similar number from P18 to P22. We estimate that about a quarter of a million rod cells are dying every day. In five days, 1.25 million rod cells would have died and this is a gross underestimate of the dead rod cells because by P28, approximately half of the rod cells present at P15 would have disappeared!