Morphological analyzes of microglia heterogeneity and dynamics during photoreceptor degeneration in vitro: presumptive dark microglia in porcine retina

In the adult retina, ramifying microglia interact with the outer plexiform layer (OPL) monitoring the synaptic integrity between photoreceptors and post-synaptic target cells. Microglia are reactive during photoreceptor diseases, but their disease-related function(s) are not fully understood. Retinal explant cultures are model systems used to study degenerative events including photoreceptor degeneration and gliosis. Our culture paradigm, with adult porcine retinas subjected to coculture with human A-retinal pigment epithelia-19 (ARPE) cells, is an experimental approach resulting in improved photoreceptor survival and reduced gliosis. Under the in vitro pathological conditions with photoreceptor degeneration, reactive Iba1-and CD11b-immunoreactive microglia and their processes positioned in proximity with the OPL and among photoreceptor outer segments. Coculture for 3 days with ARPE-cells resulted in a significantly increased density of microglia at the OPL. After 5 days of culture, the density of microglia at the OPL was similar between coculture and control specimens. Electron microscopy revealed the presence of two subtypes of microglia: one exhibiting a dark nucleus and cytosol with dilated endoplasmic reticulum, vacuoles, endosomes and mitochondrial variations. This subtype localized close to synaptic structures in the OPL. The other subtype appeared as pale phagocytic microglia localized among degenerating outer segments. The Iba1-and CD11b-immunoreactive microglia in degenerating retina may be of two separate subtypes, which differ in localization, subcellular morphology and perhaps function.