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Steven K. Fisher, Gabriel Luna, Patrick Keeley, Jiyun Byun, Benjamin E. Reese, Geoffrey P. Lewis; Astrocyte Reactivity Studied In High-Resolution Mosaics Of Detached Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6589.
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The goal of this study was to characterize normal mouse retinal astrocytes and determine how they change after retinal detachment. For this purpose we used high-resolution retinal mosaics, antibody labeling, and dye filling in mouse retinal whole mounts.
Retinal detachments (1 to 16 weeks) were created in the right eye of C57BL/6J mice; the opposite eye served as control. Retinal whole mounts were immunolabeled with anti-GFAP and anti-collagen IV (for blood vessels). In some cases, astrocytes were also filled with Lucifer yellow or Alexa 568. Images of complete whole mounts were collected as 300-500 high-resolution individual z-stacks. Projection images of the z-stacks were subsequently stitched into a full-resolution mosaic using imago software (Mayachitra, Inc., Santa Barbara, CA).
Normally, GFAP labeled astrocytes display a planar morphology with processes that are smooth and tapered in appearance making discrete focal contacts with blood vessels. After detachment they become reactive as evidenced by a more jagged, irregular appearance of the GFAP cytoskeleton and diffuse interaction with blood vessels. The reactive cells do not appear to extend processes laterally, but do show hypertrophy via extensive growth into the inner plexiform layer, usually along blood vessels. This was first observed in dye-filled cells but subsequently confirmed to be a widespread occurrence using anti-GFAP labeling.
Using large-scale imaging of mosaics allowed visualization of the entire retina at high resolution. This method does not require sampling procedures because it allows observing essentially the entire population of astrocytes along with regional variation in their structure and reactivity. Using this technique and taking advantage of the relatively less reactivity of Muller cells in the mouse retina, we were able to determine that retinal astrocytes, like homologous cells elsewhere in the CNS are highly responsive to injury.
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