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K. Hockmann, M. Duong, A.K. Ball; Reactive Microglia Are Neuroprotective Following ET–1 Mediated Injury . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4857.
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© ARVO (1962-2015); The Authors (2016-present)
Injection of endothelin–1 (ET–1) into the rat eye causes a gradual loss of retinal ganglion cells (RGCs) and their axons in the optic nerve over 4 weeks. Microglia (MG) become activated 2–7 days after ET–1 injection, preceding the detectable loss of RGCs by 1 week, suggesting that they may play a role in determining the fate of RGCs following injury. In the present study we inhibited MG activation after ET–1 treatment and examined the effect on RGC survival.
Rat RGCs were retrogradely labeled with Fluorogold (FG) from the superior colliculus 2 days prior to intraocular injection with ET–1 (5µl; 500µM). Animals were injected intraperitoneally with saline (0.5 ml; control) or minocycline (0.5ml ; 45mg/kg), an inhibitor of MG activation, 3 hours prior to ET–1 intraocular injection as well as immediately afterwards. Animals were subsequently injected with saline or minocycline once a day for 2 days after ET–1 treatment. Animals were killed 2, 4, 7, 14 and 21 days after ET–1 injection, their eyes fixed in phosphate buffered 4% formaldehyde, and processed for immunohistochemistry. RGC density was determined by counting FG immunoreactive (–IR)(Fluorochrome; 1:500) somas in confocal micrographs of flatmounted retinas. Inhibition of MG activation was assessed by examining the colocalization of Iba–1 (NIH; 1:500) and the lysosomal membrane antigen, ED–1 (Serotec; 1:250) in cryostat sections. Activated MG were differentiated from resting MG when Iba–1–IR MG also expressed ED–1.
In contrast to saline injected animals, ED–1–IR was not colocalized with Iba–1–IR MG in minocycline injected animals during the times when MG usually become activated and express ED–1 (2–7 days), or at any other timepoint. In saline injected animals significant RGC loss was first observed 7 days after ET–1 injection (17%) and gradually increased (30% loss at 21 days). In minocycline injected animals the loss of RGCs was not different from saline injected animals at 7 days, but significant at 21 days (60%). The rate of cell loss between 4 and 21 days was 44 RGCs/mm2 per day in saline injected animals, compared to 84 RGCs/mm2 per day in minocycline injected animals.
Minocycline treatment was effective in preventing MG from expressing ED–1, a marker for reactive MG. Although reactive MG have been shown to secrete a number of pro–inflammatory compounds, they also play a neuroprotective role by phagocytosing damaged cells and debris. Our finding that RGC death was greater in minocycline treated animals suggests that reactive MG have a neuroprotective role in the retina.
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