Flow cytometry for the characterization of retinal neural populations and the quantification of retinal apoptosis

Autor: Pa Tsoka, Mk Tsilimbaris
Rok vydání: 2010
Předmět:
Zdroj: Acta Ophthalmologica. 88
ISSN: 1755-375X
DOI: 10.1111/j.1755-3768.2010.4414.x
Popis: Purpose The primary purpose of this study was to evaluate the potential to quantify the different retinal neuronal populations, as also the retinal detachment-induced apoptosis in Sprague–Dawley rats, in an accurate quantitative way by using flow cytometry. Methods Retinal detachment was performed on the right eye of deeply anesthetized animals. The detachment was induced by a sub-retinal injection of sodium hyaluronate. Rats were sacrifised and the eyes were enuclated to achieve retinal dissection. Tissue dissociation was accomplished with trypsin. The cells were mechanically dissociated into a single-cell suspension. At least 100.000 cells were analyzed with a FACScalibur and FlowJo software. The primary antibodies were anti-rhodopsin against rod photoreceptors, anti-PKC against rod bipolars, anti-calbindin against horizontals, anti-ChAT against cholinergic amacrine cells and anti-MAP1 against ganglion cells. Annexin-V-FITC/Propidium Iodide was used to identify apoptosis. Results Quantification of retinal neuronal cells was possible using flow cytometry. Photoreceptors had the 53.99%, the ganglion the 7%, the bipolars the 2%, the horizontal the 4% and the cholinergic amacrine cells the 1,5% in the hole mixed retinal population. Quantification of the apoptotic rate was also possible. The early apoptotic cells was 22.4% while in the control eye was 6.28% after retinal detachment. The experiments were repeated ten times and these measurements are the mean value. Conclusion Flow cytometry can be used to quantify the apoptotic neuronal cells as well as the healthy retinal neurons. It is quick and precise and it will be very useful in future in studies in neuroprotection and quantification of apoptosis during time.
Databáze: OpenAIRE
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