Abstract
Purpose :
In the rd1 mouse, there is a loss of rod photoreceptors followed later by death of cone photoreceptors. This occurs in a central to peripheral gradient. After rod death, the RPE cells, which are normally arranged in a regular hexagonal pattern, become dysmoprhic in a similar central to peripheral gradient. We wanted to try to correlate the density of surviving cones with the level of RPE distress as measured by morphology.
Methods :
Eyes from rd1 mice were harvested and processed for whole-mount with immunostaining for cone arrestin to detect cones and phalloidin for RPE, then imaged on a spinning-disc confocal microscope. Standardized central and peripheral locations were imaged in a series of eyes with a range of ages from P40 to P127. A customized eight-step cell profiler pipeline was used to automatically segment and measure the shape of the RPE. Cones were manually counted by using Fiji to identify each individual cone arrestin positive cell. Correlation coefficients were calculated in GraphPad prism.
Results :
Qualitatively, areas with dysmorphic RPE (which generally appeared larger with loss of the regular hexagonal shape) were frequently associated with fewer cones. With age, the degree and extent of dysmoprhic RPE generally increased. As size of the RPE was qualitatively an impressive feature, we examined the correlation of mean area of the RPE in the imaged field with number of cones. Linear regression obtained an R-squared value of 0.87 with a p-value of 0.02 for central cones. In the periphery, where the RPE is less distressed, the relationship was weaker as the R-squared value was .56 with a p-value of 0.086. As aspect ratio has been found to be particularly capable of distinguishing between RPE rescued with overexpression of Nrf2 and unrescued RPE, we also examined the correlation with this parameter. Interestingly, correlation between aspect ratio and central cone counts was much weaker (R-squared of 0.16 and 0.32 respectively, p values > 0.05).
Conclusions :
Here we report qualtiative and quantitative association of increased cone loss in degeneration in areas in which the RPE has become more dysmorphic. In this small series, RPE area seems to correlate best with cone density in the central retina. Understanding the relationships between RPE health and cone density is an important part of identifying a potential contributor to photroeceptor survival in retinitis pigmentosa.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.