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P. Tsoka, M. K. Tsilimbaris; Quantification of Retinal Detachment-Induced Apoptosis by Flow Cytometry in an Experimental Rat Model. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2242.
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The primary purpose of this study was to evaluate the potentialto quantify the retinal detachment-induced apoptosis in theretina of Sprague-Dawley rats in an accurate quantitative wayby using flow cytometry.
Retinal detachment was performed on the right eye of deeplyanesthetized animals. The detachment was induced by a sub-retinalinjection of sodium hyaluronate. Rats were sacrifised at 72hours and the eyes were enuclated to achieve retinal dissection.Tissue dissociation was accomplished with trypsin. Trypsin actionwas blocked and the cells were mechanically dissociated intoa single-cell suspension by gentle pippeting. The cells thenwere incubated with Neural Cell Adhesion Molecule and finallywith annexin-V-FITC/Propidium Iodide (PI). At least 100.000cells were analyzed with a FACScalibur and FlowJo software.Cell debris were exluded from analysis by gating with forwardscatter and side scatter as indicators.
Quantification of the retinal detachment-induced apoptosis inthe neuronal cells of the retina was possible using flow cytometry.In this study the mean value of the early apoptotic cells (annexinpositive/PI negative) was 22.4% (second picture) while in thecontrol eye was 6.28% (first picture). The experiments wererepeated ten times.
Flow cytometry can be used to quantify the apoptotic neuronalcells after retinal detachment. It is quick and precise andit is also possible to study and quantify apoptosis/retinalcell type. Flow cytometry will be very useful in future in studiesin neuroprotection as also in quantification of apoptosis duringtime.
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