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Ahad M. Siddiqui, Thomas F. Sabljic, Alexander K. Ball; Down Regulation of BM88 after Optic Nerve Crush. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6577.
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BM88 (C38; Cend1) is a cell cycle exit and neuronal differentiation protein that is expressed in the rat eye by post-mitotic retinal ganglion cells (RGCs). BM88 labeling has been used as a marker of surviving RGCs after optic nerve (ON) injury. Thy1.1 has also been used as a marker for RGC loss after transection but after ON crush the decrease in thy1 expression precedes the loss of RGCs. Differences in the expression of RGC markers may depend on the type of injury. The purpose of this study was to determine if BM88 expression was correlated with RGC loss after ON crush.
Sprague Dawley rats (young adults; 225-250g) were injected with fluorogold (FG) in the superior colliculus to label RGCs and received ON crush 3 days later. The eyes were collected at 4, 7, and 14 days after injury. Control rats only received FG injections. The eyes were fixed with buffered 4% paraformaldehyde and then cryoprotected with 30% sucrose overnight. Frozen sections were labeled with rabbit α BM88 (S11; Santa Cruz; 1:100) overnight. Primary antibody was visualized with donkey α rabbit Texas Red (Jackson Immuno; 1:100). The BM88 and FG labeled RGCs were counted using an epi-fluorescence microscope. Intensity of the BM88 cell labeling was measured from images standardized for exposure using NIH ImageJ.
There was an expected decrease in FG labeled RGCs after ON crush (day 4: 19.2%, day 7: 48.9%, day 14: 71.8%). In control retinas 98.9±2.2% of BM88 immunoreactive (-IR) RGCs were co-localized with FG. Similarly, 4 days after ON crush 93.0±4.9% of BM88-IR RGCs were co-localized with FG, which was not significantly different than from control (ANOVA). However, there was a significant down regulation of BM88 by RGCs 7 days after injury. At 7 and 14 days after ON crush there were only 11.5±7.5% and 11.0±11.3% of BM88-IR RGCs that co-localized with FG. Not only were there fewer RGCs labeled with BM88 but there was also a reduction in the intensity of the remaining labeled cells. Seven days after injury the intensity of BM88 staining was reduced by 51.5% compared to staining in control retinas.
Nearly all BM88-IR RGCs co-localized with FG labeled RGCs in control retinas. However, both the number of BM88-IR RGCs and their intensity decreased gradually between 4 and 14 days, preceding the loss of FG labeled cells. These findings indicate that BM88 is not a good marker of surviving RGCs in the ON crush model.
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