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SeokHwan Kim, Juyeong Oh, YuJeong kim, Jae Hun Kim, Dae Yu kim, Ki Ho Park, Su bin Park; Optical Observation of Retinal Ganglion Cells and their Apoptosis in Optic Nerve Degeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5836.
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© ARVO (1962-2015); The Authors (2016-present)
Apoptotic loss of retinal ganglion cells (RGCs) is involved in various optic neuropathies, and its extent is closely related to visual impairment. Direct imaging and counting of RGCs is beneficial to the evaluation of RGC loss, but these are challenging in a use of conventional techniques due to RGCs transparency and hypo-reflectivity as a part of the light-transmitting structures of the retina. In this research, by using a differential interference contrast (DIC) microscopy, the direct observation of normal and apoptotic loss of RGCs as well as other neuronal cells existing in ganglion cell layer is performed.
Rat retina samples according to the time lapse of 1 day, 1 week and 2 weeks after optic nerve transection (ONT) and retrograde labeling of RGCs with fluorescein are prepared in flat mount. Morphological difference of each sample is imaged using DIC microscopy. Also, fluorescence microscopy is incorporated with DIC microscopy for the differentiation of RGCs from other cells such as amacrine cells coexisting in ganglion cell layer. The number of overall cells, ganglion cells only, and apoptotic RGCs in ganglion cell layer are counted and analyzed according to the time lapse after optic nerve transection.
Figure 1 shows the images of cells in ganglion cell layer with three different rat retina samples. RGCs are differentiated from other cells such as amacrine cells (arrowhead) by comparing DIC microscopic images (Figure 1B) with those of fluorescence microscopy (Figure 1A), in which RGCs are only affected by retrograde labeling by dextran tetramethylrhodamine. For the case of rat model, RGCs are clearly distinguishable from amacrine cells due to morphological characteristics. The number of RGCs significantly decreased over time in the ONT model: from 18.6±3.0, 16.4±1.9 and 13.6±1.7 (mean±SD, n=5) at each location (near, mid and far) in the control eyes to 0.9±0.5, 0.9±0.5 and 0.7±0.6 (mean±SD, n=5) at post-ONT 2 weeks. Apoptotic RGCs are mostly observed in 1 week after ONT.
Observation of RGCs along with other cells in ganglion cell layer is successfully performed. The experimental results show that DIC microscopy can be a possible tool for analyzing retinal diseases models ex vivo. Further studies on in-vivo retinal imaging of animal models will be performed.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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