Tsuda Satoru, Tanaka Yuji, Kunikata Hiroshi, Yokoyama Yu, Yasuda Masayuki, Ito Azusa, Nakazawa Toru
Department of Ophthalmology, Tohoku University Graduate School of Medicine, Japan.
Department of Ophthalmology, Tohoku University Graduate School of Medicine, Japan; Department of Retinal Disease Control, Tohoku University Graduate School of Medicine, Miyagi, Japan.
Exp Eye Res. 2016 May;146:179-188. doi: 10.1016/j.exer.2016.03.017. Epub 2016 Mar 21.
The retinal ganglion cells (RGCs) are the main source of therapeutic targets for neuroprotective glaucoma treatment, and evaluating RGCs is key for effective glaucoma care. Thus, we developed a minimally invasive, quick, real-time method to evaluate RGC death in mice. In this article we describe the details of our method, report new results obtained from C57BL/6J mice, and report that our method was usable in wild type (WT) and knockout (KO) mice lacking an RGC-death-suppressing gene. It used a non-invasive confocal scanning laser ophthalmoscope (cSLO) and a low molecular weight, photo-switching, cell-impermeant, fluorescent nucleic acid dyeing compound, SYTOX orange (SO). The RGCs were retrogradely labeled with Fluorogold (FG), the optic nerve was crushed (ONC), and SO was injected into the vitreous. After ten minutes, RGC death was visualized with cSLO in vivo. The retinas were then extracted and flat mounted for histological observation. SO-labeled RGCs were counted in vivo and FG-labeled RGCs were counted in retinal flat mounts. The time course of RGC death was examined in Calpastatin KO mice and wild type (WT) mice. Our in vivo imaging method revealed that SO-positive dead RGCs were mainly present from 4 to 6 days after ONC, and the peak of RGC death was after 5 days. Moreover, the number of SO-positive dead RGCs after 5 days differed significantly in the Calpastatin KO mice and the WT mice. Counting FG-labeled RGCs in isolated retinas confirmed these results. Thus, real-time imaging with SO was able to quickly quantify ONC-induced RGC death. This technique may aid research into RGC death and the development of new neuroprotective therapies for glaucoma.
视网膜神经节细胞（RGCs）是青光眼神经保护治疗的主要治疗靶点来源，评估RGCs是有效青光眼治疗的关键。因此，我们开发了一种微创、快速、实时的方法来评估小鼠RGCs死亡情况。在本文中，我们描述了我们方法的细节，报告了从C57BL/6J小鼠获得的新结果，并报告我们的方法可用于缺乏RGC死亡抑制基因的野生型（WT）和敲除（KO）小鼠。它使用了一种非侵入性共焦扫描激光检眼镜（cSLO）和一种低分子量、光开关、细胞不透性的荧光核酸染色化合物SYTOX橙（SO）。用荧光金（FG）对RGCs进行逆行标记，压迫视神经（ONC），并将SO注入玻璃体。十分钟后，用cSLO在体内观察RGCs死亡情况。然后取出视网膜并平铺进行组织学观察。在体内计数SO标记的RGCs，在视网膜平铺标本中计数FG标记的RGCs。在钙蛋白酶抑制蛋白敲除小鼠和野生型（WT）小鼠中检查RGCs死亡的时间进程。我们的体内成像方法显示，SO阳性死亡RGCs主要在ONC后4至6天出现，RGCs死亡高峰在5天后。此外，5天后SO阳性死亡RGCs的数量在钙蛋白酶抑制蛋白敲除小鼠和WT小鼠中有显著差异。在分离的视网膜中计数FG标记的RGCs证实了这些结果。因此，用SO进行实时成像能够快速量化ONC诱导的RGCs死亡。这项技术可能有助于研究RGCs死亡以及青光眼新神经保护疗法的开发。
