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Mark Magharious, Philippe M. D'Onofrio, Adam C. Hollander, Paulo D. Koeberle; Quantitative Itraq Analysis Of Retinal Ganglion Cell Degeneration After Optic Nerve Crush. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5446.
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Apoptotic Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment and can be modeled by optic nerve crush. Optic nerve crush induces the apoptotic cell death of ~70% of RGCs within the first 14 days after injury, while also permitting the study of RGC axon regeneration. The purpose of this study was to identify novel targets that are involved in RGC apoptosis, by studying global protein expression at different timepoints after optic nerve crush.
Adult female Sprague Dawley rats received a calibrated intraorbital optic nerve crush under isoflurane anesthesia. Retinas were extracted at 1, 3, 4, 7, 14 or 21 days after optic nerve crush (n=8 for each timepoint), and subjected to iTRAQ proteomics. An additional group of retinas (n=8) was used as the baseline normal. iTRAQ labeling and protein quantitation by mass spectroscopy (LC/MS/MS Analysis) was performed using an 8-plex procedure. We examined the effects of intraocular delivery of Thymosin-beta4 on RGC survival and axon regeneration after axotomy or optic nerve crush, respectively. The density of surviving, Fluorogold labeled RGCs was calculated from fixed flatmounted retinas at 14 days postaxotomy. RGC regeneration was examined at 21 days postaxotomy in longitudinal frozen sections of optic nerve stained for the axon marker GAP-43.
In total, 337 proteins were identified and quantified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated and 29 showed decreased expression (<0.7x or >1.3x) relative to normal retinas. Patterns of protein upregulation and downregulation were grouped by timepoint, permitting the identification of protein targets that are involved in RGC death. Among the differentially expressed proteins, Thymosin-beta4 showed an early upregulation at 3 days, the timepoint that immediately precedes RGC death. Intraocular injections of Thymosin-beta4 significantly increased RGC survival by ~3-fold compared to control treatment (p<0.001), and increased the mean number of regenerating axons per nerve section at three different distances beyond the lesion site (<250 micron, 250-500 micron, >500 micron; p<0.001), following optic nerve crush.
Overall, our study identified numerous proteins that are differentially regulated at key timepoints after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the diseased or injured adult retina.
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