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Micah A Chrenek, Jana Sellers, Stephanie L Foster, P Michael Iuvone, Jeffrey H Boatright; Novel method for quantifying loss of retinal ganglion cells in mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):397.
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Retinal ganglion cells (RGC) are damaged in glaucoma and other optic neuropathies such as traumatic blast injury. Current methods used to quantify RGC loss are staining RGCs in flatmounts, imaging and counting the number of surviving cells. This is a labor intensive and time consuming method. We hypothesized that a microplate reader assay could quickly quantify the number of surviving RGCs using either immunostains for RGC specific markers or mice that express cyan fluorescent protein (CFP) under a Thy1 promoter.
Thy1-CFP and control (background) C57BL/6J mice were obtained from Jackson Laboratories. Optic nerve crush was performed unilaterally in each mouse with the fellow eye serving as a control. Two weeks after crush, retinas from THY1-CFP mice were individually homogenized in RIPA buffer with protease inhibitors and homogenates plated into a black 96-well plate. Average fluorescence was measured using a fluorescent microplate reader (Synergy H1 model, BioTek). Retinal flatmounts of C57BL/6J mice were immunostained for an RGC-specific antigen (Brn3A) and immunopositive cells counted. After counting, the retinas were homogenized and fluorescence was measured using the plate reader to allow direct comparison to counts.
For Thy1-CFP retinas, average fluorescence readings (± SEM) for uncrushed and crushed retinas were 39095 ± 1507 and 23062 ± 2975 fluorescence units. This corresponds to a 41% decrease in RGC signal in the eyes that received the optic nerve crush as compared to control eyes. A similar decrease of 41% was found in RGC-specific immunopositive cell counts of C57BL/6J flatmounted retinas. Lysates of the immunostained retinas showed a 44% decrease in fluorescence readings following crush.
Given these comparable outcomes, we conclude that the fluorescent plate-reader protocol provides the same quantitative sensitivity and accuracy as conventional cell counting methods but with considerably less time and effort, as imaging and cell counting is avoided. This method could be used as a quick, informative measurement of the progression of RGC loss following optic nerve injury.
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