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Peter Heiduschka, Nan Su, Jeannette König, Annika Höhn, Tilman Grune, Constantin Uhlig, Nicole Eter; Response of murine retinal microglia towards subretinal injection of human lipofuscin. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2447. doi: https://doi.org/.
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In the aged retina, lipofuscin accumulates in the RPE and can be released to both basal and apical sides. As microglial cells may migrate into the subretinal space with increasing age, reaction of microglial cells towards lipofuscin may have important implications for AMD development and progression. In recent in vitro experiments, we found a strong inflammatory reaction of microglial cells towards lipofuscin. As a next step of our studies, we injected lipofuscin subretinally in mice to check reaction of the microglia in vivo.
Human lipofuscin was isolated from the RPE of human donor eyes. Suspension of lipofuscin was injected subretinally in eyes of adult C57BL/6J mice. Eyes were isolated at 0, 1, 3, 5, 7, 14 or 28 days after injection. Localisation and shape of microglial cells was checked by CD11b and Iba1 immunohistochemistry (IHC) in cryo sections. IHC was also performed to check the retina for VEGF, FGF-2, IL-6 and TNF-α. In additional experimental groups, the microglial inhibitors minocycline or the peptide Thr-Lys-Pro (TKP) were applied topically onto the eyes of the mice.
Injected lipofuscin could be detected in the eyes by its autofluorescence. Lipofuscin particles were found in the subretinal space as well as distributed into the outer retinal layers. Microglial cells migrated into the subretinal space, many of them displaying rounded morphology. Autofluorescent lipofuscin was not visible any more one week after injection. Immunoreactivity was increased clearly for IL-6 and TNF-α 3 to 7 days after injection and decreased afterwards, with part of microglia being positive for these cytokines. We saw no increase in immunoreactivity for FGF-2 and VEGF. Treatment with microglial inhibitors resulted in a decelerated removal of lipofuscin particles and a clearly decreased levels of IL-6 and TNF-α, whereas migration into subretinal space not appears to be impaired.
Microglial cells react quickly when lipofuscin is injected subretinally in mouse eyes by migration into subretinal space and increased levels of inflammatory cytokines IL-6 and TNF-α. As we saw no enhanced production of the growth factors VEGF and FGF-2, it may be questioned if lipofuscin promotes neovascularisation, and major source of stress connected with enhanced load of lipofuscin may be inflammatory processes. Importance of slower lipofuscin scavenging for retinal pathology still has to be clarified.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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