Rat retinal ganglion cell loss caused by kainate, NMDA and ischemia correlates with a reduction in mRNA and protein of Thy-1 and neurofilament light.
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.
Chidlow
Glyn
G
Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, OX2 6AW, Oxford, UK. glyn.chidlow@eye.ox.ac.uk
Osborne
Neville N
NN
D000818:Animals; D015153:Blotting, Western; D018690:Excitatory Amino Acid Agonists / Q000008:administration & dosage / Q000633:toxicity*; D005123:Eye / Q000098:blood supply*; D007150:Immunohistochemistry; D007511:Ischemia / Q000473:pathology*; D007608:Kainic Acid / Q000008:administration & dosage / Q000633:toxicity*; D007700:Kinetics; D008297:Male; D008845:Microinjections; D016202:N-Methylaspartate / Q000008:administration & dosage / Q000633:toxicity*; D016900:Neurofilament Proteins / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018800:Thy-1 Antigens / Q000096:biosynthesis*; D014822:Vitreous Body / Q000502:physiology
D018690:Excitatory Amino Acid Agonists; D016900:Neurofilament Proteins; D012333:RNA, Messenger; D018800:Thy-1 Antigens; D016202:N-Methylaspartate; D007608:Kainic Acid