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F.W. Fitzke, V. Tsatourian, L. Guo, A. Maass, V. Luong, M.F. Cordeiro; Time–Course of Single Cell Apoptosis in vivo Using Video and Image Analysis of Retinal Ganglion Cell Disease Model . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4823.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Apoptosis is a dynamic process of variable length depending on the cell type, nature of the inducing agent and the measured parameter. Continuous monitoring of single cell apoptosis has previously only been possible in tissue culture, with fluorescence and video microscopy, scanning and flow cytometry, and microculture kinetic assays. Using our newly developed technique of real–time imaging of retinal ganglion cell apoptosis, we analysed the time course of single retinal ganglion cell (RGC) apoptosis induced by staurosporine. Methods: Using a variety of agents to induce neuronal apoptosis, 20 DA rats underwent general anaesthetic following which optimal doses of the apoptotic inducer (e.g. staurosporine, okadeic acid) and fluorescent–labeled annexin 5 were administered intravitreally. Rat eyes were then imaged using confocal scanning laser ophthalmoscopy (cSLO), as previously described. Videos of scanned retinal areas were recorded and images assessed for fluorescence. Different individually–labelled cells were identified on the video and analysed over a period of 5 hours. Each identified cell was studied in the sequential single frames (each comprising 768 x 576 pixels) making up the video (25 frames/sec) and its average pixel value was calculated over the whole 5 hour time period. Results: Video sequences of the same scanned areas with the cSLO, showed a random pattern of distribution and appearance of fluorescent cells throughout the retina. Graphs were constructed showing the fluorescence profile over time for each individual cell. Cells were seen to fluoresce from 30 minutes to 4 hours from the first observation of apoptosis. The average duration of a single cell apoptosing, as judged by the duration of annexin 5 labeling and the pattern of fluorescence over time, was 60 minutes, ranging from 30 to 100 minutes. Conclusions: This method of analysis has allowed us to delineate the temporal sequence of apoptosis taking place in individual cells in vivo. We demonstrate asynchronous involvement of RGC cells apoptosing throughout the retina, with the whole process lasting 1 to 2 hours. This is analogous to the morphological analysis of apoptosis in vitro, which suggests a 2 hour period between the appearance of plasma membrane protrusions to the shedding of blebs of apoptotic cells. Annexin 5 detects apoptosis significantly earlier than other methods. This has great implications for our technique being used to assess potential therapies preventing early apoptosis e.g. caspase inhibitors.
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