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A.K. Ball, K. Hockmann, M. Duong; Proliferation Of Microglia Following ET–1 Mediated Retinal Ganglion Cell Death . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4833.
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Following treatment with ET–1, 40% of retinal ganglion cells (RGCs) are gradually lost over a 4 wk period. Glial reactions in astrocytes, Muller Cells, and microglia (MG) preceed the loss of RGCs, which is first detectable at 2 wks. Changes in MG occur within 2 days and peak 4 days after ET–1 treatment. These changes include retraction of their ramified processes, migration, and expression of the lysosomal antigen ED–1. The increase in MG numbers observed in the ganglion cell layer could be due to migration from other layers or proliferation. In the present study we examined proliferation of MG using BrdU immunohistochemistry.
ET–1 (5µL; 500µM) was injected into the vitreous chamber of adult rat eyes. Immediately after ET–1 administration, animals were given intraperitoneally–administered 5–bromo2’–deoxy–uridine (BrdU)(1ml; 25µg/µl). BrdU was also administered in the drinking water throughout the timecourse of the study. Animals were killed at 2, 4, 7, 14 and 21 days post–ET–1 injection, eyes were fixed in phosophate buffered 4% formaldehyde and processed for double label immunohistochemistry. MG were identified using polyclonal antisera directed against Iba–1 (NIH; 1:500) and astrocytes were identified using antisera directed against GFAP (Chemicon; 1:1000). GFAP and Iba–1 immunoreactivity (–IR) was colocalized with BrdU immunoreactive cells (monoclonal; Chemicon; 1:1000) using confocal microscopy to identify the cells that had incorporated BrdU.
BrdU–IR was not detected in control retinas that had not received ET–1 treatment. BrdU–IR nuclei were first detected in retinas 2 days after ET–1 treatment and labeling was extensive in retinas fixed 4 and 7 days after ET–1 treatment. The number of BrdU–IR nuclei were significantly fewer in retinas after14 days and rare in retinas 21 days after ET–1 treatment. BrdU–IR nuclei were located in three distinct retinal layers: the nerve fiber layer (NFL), proximal inner plexiform layer (IPL) and distal IPL. These layers correspond to the location of astrocytes (NFL) and microglia (proximal and distal IPL). Few BrdU–IR nuclei were colocalized with Iba–1–IR cells 4 days after ET–1 treatment. There was an increase in colocalization between BrdU and Iba–1 after 7 and 14 days post ET–1 injection.
These studies suggest that MG proliferate, as well as migrate in response to ET–1 mediated RGC loss. MG proliferation begins when they first show signs of activation and continues until they begin to return to their resting state. It is unknown why MG activation and proliferation occurs between 4 and 7 days and then returns to control levels despite the continued loss of RGCs.
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