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Cheri Stowell, An Zhou, George A. Cioffi, Claude F. Burgoyne; Down Regulation Of Myelin Proteins In The Myelination Transition Zone (mtz) In The Non-human Primate (nhp) Early Experimental Glaucoma (eeg) Model. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3839.
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We have previously reported profound optic nerve head (ONH) connective tissue deformation and remodeling in NHP EEG. Here we detect proteomic changes in retrolaminar MTZ myelin proteins in NHP EEG and seek to validate them by western blotting (WB) and immunohistochemistry (IHC).
Following initiation of unilateral, laser-induced, chronic IOP elevation, 10 NHPs were sacrificed at the onset of reproducible ONH surface change (EEG definition) for either proteomic analysis by mass spectrometry (MS) followed by WB or IHC. Based on the magnitude of post-laser IOP elevation, each animal was retrospectively assigned to either a mild IOP (MIOP EEG - peak IOP less than 25 mm Hg, n= 4) or high IOP (HIOP EEG - peak IOP greater than 30 mm Hg, n=6) group. ONH trephines of either 6 mm (for MS and WB analyses; 2 mild and 2 high IOP animals) or 10 mm (for IHC; 2 mild and 4 high IOP animals) were taken from both eyes of each animal. For MS analyses, proteins extracted from individual trephines were trypsin digested and analyzed with an LTQ ion-trap MS system (Thermo Finnegan) with 3 technical replicates of each sample. Using a MS spectral counting approach, ratios of spectral counts for proteins in the EEG eye of each animal compared to its contralateral normal eye were determined. For IHC, individual trephines were fixed, paraffin embedded, sectioned at 5um and analyzed with IHC.
MS analysis detected a significant decrease for both 2’, 3’-cyclic nucleotide 3’ phosphodiesterase (CNPase) and myelin basic protein (MBP) in the HIOP EEG eyes (average ratios -1.87 and -1.57 respectively; p-values < 0.001, GEE analysis), and a moderate decrease in the mild IOP EEG eyes (not statistically significant) in the MIOP EEG eyes. Results of semi-quantitative WB analyses demonstrated reduced levels of both proteins in both the HIOP and MIOP EEG eyes compared to that in control eyes (CNPase ratios -1.20 HIOP and -1.16 MIOP; MBP ratios -1.75 HIOP and -1.72 MIOP). Results of IHC with an anti-human CNPase or an anti-MBP antibody revealed consistent down regulation of both proteins within the retrolaminar MTZ in all MIOP and HIOPeyes compared to their control eyes.
In NHP EEG, axon myelination protein alterations are present within the retrolaminar MTZ. The timing of these alterations and their relationship to ONH axon homeostasis, connective tissue remodeling and astrocyte, microglial and oligodendrocyte activity remains to be determined.
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