Quantifying Photoreceptor Cell Loss in Mouse Retina

Mouse eyes were enucleated, processed, embedded, sectioned, and hematoxylin and eosin (H&E) stained by Excalibur Pathology (Norma, OK)^{51 (link)}. H&E-stained sections were imaged on an Axio Scan.Z1 Slide Scanner equipped with a Hitachi HV-F203 camera and a Plan Apo 20×/0.8-NA objective (Carl Zeiss Microscopy, White Plains, NY). The number of nuclei spanning the outer nuclear layer was counted manually on both the superior and inferior regions of the retina at various distances from the optic nerve. Three different sections from the same eye were quantified and averaged. To determine the kinetics of photoreceptor cell loss, values from 600, 800, and 1000 μm from the optic nerve were averaged and plotted. Data were fit with an equation for one-phase decay ( $y=\left(y_0-plateau\right)\times e^{-kx}+plateau$ ) using non-linear regression to obtain the rate constant (k) using Prism 9 (GraphPad Software, San Diego, CA). The variable y₀ was set to be common among data sets analyzed together and the plateau was fixed to equal 1 to exclude the loss of cone photoreceptor cells.