Imaging and Differentiation of Retinal Ganglion Cells in Ex Vivo Experimental Optic Nerve Degeneration by Differential Interference Contrast Microscopy

Purpose: 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 processes are challenging with the conventional techniques, due to the transparency and hypo-reflectivity of RGCs as light-transmitting structures of the retina. Differential interference contrast (DIC) microscopy, which can provide real-time images of transparent specimens, is utilized to image neuronal cells including RGCs in the ganglion cell layer (GCL). Methods: Herein, we show that the neuronal cells within each GCL in an explanted rat retina, including the inner nuclear layer and the outer nuclear layer, can be imaged selectively by transmission-type DIC microscopy. RGCs were also differentiated from non-RGCs by the objective method. Results: RGCs were differentiated from non-RGCs in the GCL by their morphological features on DIC images with the aid of retrograde fluorescence labeling. Loss of RGCs was detected in optic-nerve-transection and retinal-ischemia-reperfusion models by DIC imaging. The images obtained from the reflection-type DIC microscopy were comparable to those from the transmission-type DIC microscopy. Conclusions: This method enables direct optical visualization of RGCs in experimental optic-nerve degeneration, thus providing the opportunity for more accurate evaluation of optic neuropathies as well as more effective investigation of diseases.