Assessing retinal ganglion cell death and neuroprotective agents using real time imaging.
Assessing retinal ganglion cell death and neuroprotective agents using real time imaging.
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使用实时成像评估视网膜神经节细胞死亡和神经保护剂。
DOI：
10.1016/j.brainres.2019.02.008
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Nakazawa T.
中科院分区：
文献类型：
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作者：
Ito A;Tsuda S;Kunikata H;Toshihumi A;Sato K;Nakazawa T.
视网膜神经节细胞(RGC)死亡的评估是视网膜疾病治疗的关键部分。此前，我们使用了一种基于Sytox Orange(SO)的实时成像方法来评估接受视神经挤压的小鼠的视网膜节细胞。在这里，我们使用N-甲基-D-天冬氨酸(NMDA)损伤的大鼠来验证我们的模型，并评估神经保护剂对RGC的影响。大鼠接受NMDA损伤和玻璃体内注射SO，SO是一种针对核酸的细胞无害的染色化合物。10分钟后，无创共焦扫描激光眼底镜可显示受损或死亡的细胞。最后，采用荧光金逆行标记和Alexa荧光素V-Annexin V(Annexin V)染色，对视网膜进行平铺，以确认RGC死亡。实时成像显示，SO阳性细胞在注射NMDA后2 h开始显著增加，3 h接近平台期，荧光金和Annexin V阳性细胞在视网膜中呈阳性反应。此外，与羧甲基纤维素相比，SNJ-1945治疗后SO阳性视网膜细胞的数量显著减少。这些结果在分离的视网膜中得到了证实。因此，SO的实时成像可以快速定量NMDA诱导的RGC损伤和死亡，并评估神经保护剂。这项技术可能有助于研究开发新的神经保护疗法。
The evaluation of retinal ganglion cell (RGC) death is a key part of retinal disease care. Previously, we used a Sytox Orange (SO)-based real-time imaging method to assess the RGCs in mice that underwent optic nerve crush. Here, we used N-methyl-D-aspartate (NMDA) injury in rats to confirm our model and assess the effect of neuroprotective agents on RGCs.The rats received NMDA injury and the intravitreal injection of SO, a cell-impermeant dyeing compound that targets nucleic acid. After ten minutes, non-invasive confocal scanning laser ophthalmoscopy visualized damaged or dying cells. Finally, the retinas were flat-mounted for histological confirmation of RGC death, with retrograde Fluorogold labeling and Alexa Fluor 488 Annexin V-conjugate (Annexin V) staining. This also revealed the time course of retinal cell death and the neuroprotective effect of SNJ-1945.Real-time imaging showed that SO-positive cells significantly increased starting 2 h after NMDA injection and reached an approximate plateau at 3 h. SO-positive cells were positive for Fluorogold and Annexin V in the isolated retinas. Moreover, the number of SO-positive retinal cells was significantly lower after treatment with SNJ-1945, compared to carboxymethyl cellulose. These results were confirmed in the isolated retinas.Thus, real-time imaging with SO allows the quick quantification of NMDA-induced RGC damage and death, and evaluation of neuroprotective agents. This technique may aid research into the development of new neuroprotective therapies.