3D-imaging reveals novel organisational insights into the mouse outer retina

The retinal pigment epithelium (RPE) maintains the light-sensitive photoreceptors of the retina and forms the outer blood-retinal-barrier. Amongst its functions, RPE cells internalise and degrade discarded outer segments (OS) from overlying photoreceptors as part of the daily photoreceptor renewal. Pathogenic changes to the RPE monolayer and its underlying Bruch’s membrane (BrM) are linked with irreversibly blinding conditions including age-related macular degeneration (AMD), which is the most common cause of sight-loss in the UK. The current understanding of RPE anatomy and adjacent tissues in the outer retina is largely derived from conventional 2D-electron microscopy studies. Here, we used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central retina of C57BL/6 mice (n=3 eyes from three animals). 3D-reconstructed OS revealed larger bi-nucleate RPE cells to support more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE maintaining fewer photoreceptors. However, irrespective of their nuclei numbers, RPE cells supported larger numbers of photoreceptors than previously thought. 3D-data enabled us to calculate the angle and surface area of contact between apical RPE microvilli and interdigitating OS. 3D-reconstructed mitochondria revealed novel organisational details of these organelles in healthy RPE cells. Bi-nucleate RPE contained more mitochondria, which appeared to be fragmented. Bi-nucleate cells also contained large sub-RPE spaces that supports a likely association with pathogenic changes in the RPE-BrM interface linked with age and AMD. Use of macros quantified cell-cell interactions in the RPE monolayer. 3D-information also enabled the reconstruction of a user-manipulable virtual RPE cell and to print RPE cells in 3D. The use of perfusion-fixed tissues ensured the highest possible standard of preservation and a benchmark for comparing diseased retinal tissues from patients with AMD and other retinopathies. Our discoveries pave the way for further 3D-scrutiny of RPE anatomy, so pathogenic changes linked with age and retinopathy can be better understood. Ethics statement: Animal studies were approved by the local Ethical Research Committee (Home Office licence #P395C9E5F) and carried out in accordance with the UK Animal (Scientific Procedures) Act of 1986. Experiments conformed to the ARVO statement for the use of animals in Ophthalmic and Vision Research.