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Linnea T Taylor, Karin M M Arner, Fredrik K Ghosh; Pre-gliotic Müller cell responses in the isolated adult rat retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4471.
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Upregulation of GFAP intermediate filaments in Müller cells is a well known consequence of retinal insult. However, the timeline of glial-related events preceeding this reaction and their relation to cell death progression have not been extensively explored.
Full-thickness retinal sheets were isolated from adult rat eyes. 6x6 mm retinal explants were cultured for 1h, 3h, 6h, 12h, 24h, 48h and 5 days in vitro (DIV) using a previously established protocol with the photoreceptors positioned against the culture membrane. Adult rat eyes fixed immediately after enucleation were used as a baseline in vivo control. The grafts were analyzed morphologically using hematoxylin and eosin staining (H&E), immunohistochemistry with antibodies directed against several Müller cell proteins, and apoptosis (TUNEL labeling).
Already 1h after explantation, Müller cell expression of carbonic anhydrase II and glutamine synthetase (GS) was found to be strongly upregulated compared to baseline. Early events also included cellular retinaldehyde-binding protein (CRALBP) upregulation after 12h. These three Müller cell related proteins as well as basic fibroblast growth factor (bFGF) were progressively downregulated after 12h - 5 DIV compared to baseline whereas GFAP expression was unchanged from 1h-24h, and upregulated late, at 2-5 DIV. TUNEL labeling was evident in the ganglion cell layer (GCL) at 1h. At 6 and 12h, numerous cells were labeled in the GCL and the inner nuclear layer (INL). At 24h, scattered cells were labeled in the INL. After 48h, isolated labeled cells were found in the INL and outer nuclear layer, and at 5 DIV, a multitude of labeled cells were present in all cell layers.
Several Müller cell proteins important for retinal homeostasis alter their expression almost immediately after retinal insult. These changes preceed GFAP upregulation which is a comparably late event. The pre-gliotic events and their relation to cell death dynamics provides important insight into early cell death mechanisms after retinal injury.
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