Real-time imaging of RGC death with a cell-impermeable nucleic acid dyeing compound after optic nerve crush in a murine model

被引:19
作者
Tsuda, Satoru [1 ]
Tanaka, Yuji [1 ,4 ]
Kunikata, Hiroshi [1 ,2 ]
Yokoyama, Yu [1 ]
Yasuda, Masayuki [1 ]
Ito, Azusa [1 ]
Nakazawa, Toru [1 ,2 ,3 ]
机构
[1] Tohoku Univ, Grad Sch Med, Dept Ophthalmol, 1-1 Seiryo Machi, Sendai, Miyagi 980, Japan
[2] Tohoku Univ, Grad Sch Med, Dept Retinal Dis Control, 1-1 Seiryo Machi, Sendai, Miyagi 980, Japan
[3] Tohoku Univ, Grad Sch Med, Dept Adv Ophthalm Med, 1-1 Seiryo Machi, Sendai, Miyagi 980, Japan
[4] RIKEN, ACCC, Tsurumi Ku, 1-7-22 Suehiro Cho, Yokohama, Kanagawa 2300045, Japan
关键词
In vivo imaging; Retinal ganglion cells; Cell death; Glaucoma; Fluorescent probe; Neuroprotection; RED FLUORESCENT PEPTIDE; OPEN-ANGLE GLAUCOMA; IN-VIVO; AUTOMATED PERIMETRY; COHERENCE TOMOGRAPHY; MEMBRANE-PERMEANT; APOPTOSIS; CALPAIN; CASPASE; DAMAGE;
D O I
10.1016/j.exer.2016.03.017
中图分类号
R77 [眼科学];
学科分类号
100212 ;
摘要
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. (c) 2016 Elsevier Ltd. All rights reserved.
引用
收藏
页码:179 / 188
页数:10
相关论文
共 51 条
[1]   A QUALITATIVE COMPARISON OF THE REACTIONS OF RETINAL GANGLION-CELL AXONS TO OPTIC-NERVE CRUSH IN NEONATAL AND ADULT MICE [J].
ALLCUTT, D ;
BERRY, M ;
SIEVERS, J .
DEVELOPMENTAL BRAIN RESEARCH, 1984, 16 (02) :231-240
[2]  
Alward WL, 1998, AM J OPHTHALMOL, V126, P498
[3]  
AUTZEN T, 1990, ACTA OPHTHALMOL, V68, P327
[4]   Single-cell imaging of retinal ganglion cell apoptosis with a cell-penetrating, activatable peptide probe in an in vivo glaucoma model [J].
Barnett, Edward M. ;
Zhang, Xu ;
Maxwell, Dustin ;
Chang, Qing ;
Piwnica-Worms, David .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2009, 106 (23) :9391-9396
[5]   Synthesis and characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis [J].
Bullok, K ;
Piwnica-Worms, D .
JOURNAL OF MEDICINAL CHEMISTRY, 2005, 48 (17) :5404-5407
[6]   Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis [J].
Bullok, Kristin E. ;
Maxwell, Dustin ;
Kesarwala, Aparna H. ;
Gammon, Seth ;
Prior, Julie L. ;
Snow, Margaret ;
Stanley, Sam ;
Piwnica-Worms, David .
BIOCHEMISTRY, 2007, 46 (13) :4055-4065
[7]  
Chan SL, 1999, J NEUROSCI RES, V58, P167, DOI 10.1002/(SICI)1097-4547(19991001)58:1<167::AID-JNR16>3.3.CO
[8]  
2-B
[9]   Practical recommendations for measuring rates of visual field change in glaucoma [J].
Chauhan, B. C. ;
Garway-Heath, D. F. ;
Goni, F. J. ;
Rossetti, L. ;
Bengtsson, B. ;
Viswanathan, A. C. ;
Heijl, A. .
BRITISH JOURNAL OF OPHTHALMOLOGY, 2008, 92 (04) :569-573
[10]   Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression [J].
Chen, Xi ;
McCuef, Hannah V. ;
Wong, Shi Quan ;
Kashyap, Sudhanva S. ;
Kraemer, Brian C. ;
Barclay, Jeff W. ;
Burgoyne, Robert D. ;
Morgan, Alan .
MOLECULAR NEURODEGENERATION, 2015, 10