OS ultrastructure is substantially improved by increased Rds expression
Finally, we analyzed NMP nanoparticle-mediated structural rescue of photoreceptors in the rds +/− retina, using both light and electron microscopy, at PI-30 and PI-120, in comparison with uninjected controls. Photoreceptors in the rds +/− retina typically exhibit very short OSs with misaligned and whorl-like disc membranes. At PI-30, there was a modest increase in outer nuclear layer (ONL) thickness (Fig 5A, top), and many individual OSs exhibit improved ultrastructure (arrows, Figure 5A, bottom). By PI-120, however, virtually all photoreceptors examined showed noticeable structural improvement (Figure 5B). Consistent with the ERG results (see Figure 4), structural rescue was more pronounced in the IRBP-NMP-injected eyes compared to CBA-NMP-injected eyes at PI-120, but both exhibited OSs with orderly stacks of disc membranes.
Figure 5  Transferred NMP leads to structural rescue of the rds +/− phenotype.
Light micrographs (top row) and electron micrographs (bottom row, N = 3–5 animals per group) from rds +/− were examined. (A) At PI-30, moderate ultrastructural rescue is detected in the OSs of nanoparticle injected eyes (arrows). (B) By PI-120 significant ultrastructural improvement in OSs of nanoparticle injected eyes is apparent. OS discs are properly aligned and flattened and OS do not exhibit the swirl-like structures typical of the rds +/−. RPE, retinal pigment epithelium; OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer. Scale bar, 10 µm. In order to determine the extent of these structural improvements, we undertook a series of morphometric analyses of the nanoparticle injected-eyes compared to controls. Histological images were collected from each eye at 200 µm, 400 µm, and 600 µm from the optic nerve head (both temporally and nasally) and vertical rows of ONL nuclei and OS thickness were measured. Figure S4 shows results from two representative experimental animals, with the average values (± standard deviation) obtained from uninjected control eyes shaded in gray (accompanying supplemental methods found in Text S1). At PI-30, injected animals showed little or no increase in the number of rows in the ONL (top panels), but a definite increase in OS thickness (bottom panels). At PI-120, there was no morphometric evidence of any histological benefit from the CBA-NMP nanoparticles, while IRBP-NMP nanoparticles led to a slight increase in both the number of rows in the ONL and in OS thickness. The increase in ONL rows at PI-120 is particularly relevant, since it suggests that nanoparticle-mediated increases in RDS may slow photoreceptor cell death in the rds +/−.