Chidlow Glyn, Osborne Neville N
Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, OX2 6AW, Oxford, UK.
Brain Res. 2003 Feb 14;963(1-2):298-306. doi: 10.1016/s0006-8993(02)04052-0.
Quantification of retinal ganglion cell (RGC) loss/survival following a defined insult to the retina is a prerequisite in order to allow a comparison to be made between the effectiveness of potential neuroprotective drugs. The purpose of the present study was to extend the characterisation of our previously published semiquantitative RT-PCR assay to assess RGC loss/survival. Comparisons were made between the total mRNA levels of the ganglion cell-specific markers Thy-1 and neurofilament light (NF-L) in the retina at specific times after an intravitreal injection of N-methyl-D-aspartate (NMDA) or kainate or after 45 min of ischemia/reperfusion and also between the levels of NF-L mRNA and protein at various times after NMDA injection. Changes in Thy-1 and NF-L immunoreactivities were also observed. NMDA, kainate and ischemia/reperfusion all caused a reduction in the retinal content of Thy-1 and NF-L mRNAs and immunoreactivities. An excellent correlation was observed between the levels of the two mRNAs after these treatments. After NMDA, loss of NF-L mRNA was shown to precede loss of NF-L protein but total loss of each marker was similar after 7 days. The results of the study demonstrate that injury and subsequent death of RGCs, which occurs after ischemia/reperfusion and after intraocular injection of NMDA or kainate, can be followed by measurement of total retinal levels of Thy-1 and NF-L mRNAs and NF-L protein. The assays provides accurate, practical and complementary methods for assessing the potential benefits of neuroprotective drugs on RGCs which have been injured by a variety of experimental modalities.
为了能够比较潜在神经保护药物的有效性，对视网膜进行特定损伤后定量视网膜神经节细胞（RGC）的损失/存活情况是一项先决条件。本研究的目的是扩展我们之前发表的半定量RT-PCR检测方法的特性，以评估RGC的损失/存活情况。在玻璃体内注射N-甲基-D-天冬氨酸（NMDA）或海藻酸后特定时间，或在缺血/再灌注45分钟后，比较视网膜中神经节细胞特异性标志物Thy-1和神经丝轻链（NF-L）的总mRNA水平，同时也比较NMDA注射后不同时间NF-L mRNA和蛋白的水平。还观察了Thy-1和NF-L免疫反应性的变化。NMDA、海藻酸和缺血/再灌注均导致视网膜中Thy-1和NF-L mRNA含量及免疫反应性降低。这些处理后两种mRNA水平之间观察到极好的相关性。NMDA注射后，NF-L mRNA的损失先于NF-L蛋白的损失，但7天后每种标志物的完全损失相似。研究结果表明，在缺血/再灌注以及眼内注射NMDA或海藻酸后发生的RGC损伤和随后的死亡，可以通过测量视网膜中Thy-1和NF-L mRNA以及NF-L蛋白的总水平来追踪。这些检测方法为评估神经保护药物对因各种实验方式而受损的RGC的潜在益处提供了准确、实用且互补的方法。
