Quantitative profiling of regional protein expression in rat retina after partial optic nerve transection using fluorescence difference two‑dimensional gel electrophoresis

Abstract

To examine the difference between primary and secondary retinal ganglion cell (RGC) degeneration, the protein expression at four regions of retina including superior, temporal, inferior and nasal quadrant in a rat model of partial optic nerve transection (pONT) using 2-D Fluorescence Difference Gel Electrophoresis (DIGE) were investigated. Unilateral pONT was performed on the temporal side of optic nerves of adult Wistar rats to separate primary and secondary RGC loss. Topographical quantification of RGCs labeled by Rbpms antibody and analysis of axonal injury by grading of optic nerve damage at 1 week (n=8) and 8 weeks (n=15) after pONT demonstrated early RGC loss at temporal region, which is considered as primary RGC degeneration and progressing RGC loss at nasal region, which is considered as secondary RGC degeneration. Early protein expression in each retinal quadrant (n=4) at 2 weeks after pONT was compared with the corresponding quadrant in the contralateral control eye by DIGE. For all comparisons, 24 differentially expressed proteins (>1.2-fold; P<0.05; ≥3 non-duplicated peptide matches) were identified by mass spectrometry (MS). Interestingly, in the nasal retina, serum albumin and members of crystallin family, including αA, αB, βA2, βA3, βB2 and γS indicating stress response were upregulated. By contrast, only αB and βA2 crystallin proteins were altered in temporal quadrant. In the superior and inferior quadrants, βB2 crystallin, keratin type I, S-arrestin and lamin-B1 were upregulated, while heat shock cognate 71 kDa protein and heterogeneous nuclear ribonucleoproteins A2/B1 were downregulated. In summary, the use of DIGE followed by MS is useful to detect early regional protein regulation in the retina after localized optic nerve injury.

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics

“…The density of RGCs in the temporal and nasal quadrant after pONT was lower than their corresponding quadrants in the control eyes. Similarly to our previous report [20], the current findings indicate that primary RGC degeneration occurs in the temporal quadrant, whereas secondary RGC degeneration happens in the nasal quadrant. Loss of RGCs and glial reactivity two weeks after pONT.…”

“…This proteomics perspective using the rat pONT model contributes considerably to the understanding of the temporal and spatial aspects of progressive RGC degeneration, because of its clear separation between primary and secondary RGC degeneration in a system-wide, unbiased investigation. Consistent with previous reports [15][16][17][18][19][20], immunohistochemical techniques with antibodies against RBPMS (RGC marker) and GFAP (glial marker) demonstrated a clear separation of extensive neuronal loss in the region with primary RGC degeneration (temporal) and mild loss in the area of secondary RGC degeneration (nasal) after pONT. In contrast, increased glial reactivity was uniform over the entire retina.…”

“…Although their gene or protein regulation is under the influence of optic nerve damage [28], they are widely used as RGC markers for quantitative analysis in RGC degeneration. The present study demonstrated that the reduced levels of some structural proteins, including members of the neurofilament family (NF-L, NF-M and NF-H) and Thy-1, in the temporal retina, as well as their spatial pattern of protein expression, were consistent with the topographical quantification of RGC loss [20].…”

“…A partial optic nerve transection (pONT) model in rats has been developed to distinguish secondary degeneration from primary RGC degeneration morphologically [15][16][17][18]. The primary lesion in the optic nerve not only displays demyelination, gliosis, and axonal loss [19] but also dramatic loss of RGC bodies at one to two weeks in the retinal area corresponding to the location of the partial optic nerve injury site [20]. After weeks or months, the gradual loss of RGC bodies occurs at locations beyond the initial injury site, which is known as secondary RGC degeneration.…”

“…Therefore, we utilized a rat model of pONT with previously characterized RGC loss, then divided each retina into four retinal quadrants for probing the proteome profiles specific to primary and secondary RGC degeneration. Our previous work with fluorescence difference twodimensional gel electrophoresis successfully demonstrated regional changes in protein expression in various retinal locations after localized optic nerve injury [20]. Modern liquid chromatography-mass spectrometry (LC-MS) offers a combination of exploratory protein identification with increased sensitivity, specificity, and validations for both protein quality and quantity.…”