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Giedrius Kalesnykas, Ericka Oglesby, Mary E. Pease, Harry A. Quigley; A New Semi-automated Analysis Of Neuronal Morphology. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5364.
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© ARVO (1962-2015); The Authors (2016-present)
To develop detailed evaluation of neuronal morphology in retinal ganglion cells (RGCs) from retinal whole-mounts.
Retinal whole-mounts of transgenic mice that express yellow fluorescent protein (YFP) in a small subset of RGCs were used (strain B6.Cg-Tg(Thy1-YFPH)2Jrs/J, The Jackson Laboratory, Bar Harbor, Maine). Individual YFP-positive cells were imaged using confocal microscopy. The analysis was done using MetaMorph Offline version 188.8.131.52 image analysis software (Molecular Devices, Downington, PA). The automated macro contains both a proprietary neurite outgrowth measurement program and our specially-designed Sholl rings’ template. The results of neurite outgrowth analysis provide data such as the cell soma area, total length of outgrowth, number of outgrowths that connect to cell soma, number of branches of all the processes, mean, median and maximum process length. A Sholl analysis gives quantitative data on axon length and branching complexity in 10 concentric rings with radii of 50 to 500 µm centered on the RGC soma.
Thirty-two different YFP positive cells were analyzed from three eyes of three different mice. The mean area of YFP positive cell soma was 697±210.4 µm2 (mean±SD), the total length of skeletonized outgrowth was 3098±1442.7 µm, the mean process length was 520.4±302.5 µm, the median process length was 230.9±175.9 µm, and the maximum process length was 1743.4±1173.4 µm. There were 6.4±1.8 outgrowths that connected to cell soma and 155±76.8 branches of all the outgrowths. A Sholl analysis showed that dendritic complexity is the highest at radii of 150-200 µm from the center of RGC soma. On average the analysis of one cell took less than a minute.
A new semi-automated analysis is time-efficient and an unbiased tool to evaluate sparsely labeled neuronal morphology. We used this method to show differences in RGC morphology that were induced by experimental IOP elevation in mice (Oglesby et al., abstract submitted, ARVO 2011).
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