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E. M. McElnea, B. D. Quill, N. G. Docherty, D. M. Wallace, M. Irnaten, M. Farrell, C. J. O'Brien; Evidence of Mitochondrial Dysfunction and Oxidative Stress in Glaucomatous Human Lamina Cribrosa Cells: Lipofuscin Accumulation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6112.
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Alterations in the phenotype of glial cells of the lamina cribrosa (LC) are implicated in extracellular matrix remodelling at the optic nerve head (ONH) in glaucoma. Lipofuscin, an intralysosomal, non-degradable, autofluorescent macromolecule, accumulates in the peri-nuclear region of cells with oxidative stress induced elevations in mitochondrial turnover. Our study aimed to compare levels of lipofuscin-like material in lamina cribrosa cells from normal donor eyes (NLC) and glaucomatous donor eyes (GLC).
Post-confluent cultures of NLC and GLC were examined by transmission electron microscopy and the number and size of peri-nuclear lysososomes per high powered field (x 20,000) recorded. Cells were also stained with Sudan Black B, to assess peri-nuclear lipophilic body number and size. Peri-nuclear lysosomes were examined by live cell fluorescence microscopy and cellular autofluorescence quantified using flow cytometry (emission at 563-607nm).
The number of peri-nuclear lysosomes was increased in GLC (11.1 +/- 3.8 v 4.2 +/- 3.7, p = 0.002). A similar observation was made using Sudan Black B staining on number ( 22.10 +/- 3.57 v 13.77 +/- 5.66, p = 0.07) and size ( 2023.6 +/-611.23 v 862.8 +/- 74.23, p= 0.04 ). Perinuclear lysosomes were found to be autofluorescent and an increase in whole cell autofluorescence was observed in GLC ( 83062 +/- 45.1 v 41.01 +/- 3.9, p = 0.2).
We present evidence of increased lipofuscin formation being characteristic of lamina cribrosa cells derived from glaucomatous donors. The persistence of this phenomenon in vitro suggests that oxidative stress and alterations in mitochondrial function are important in ONH remodelling in glaucoma. Future anti-glaucoma strategies may include attempts at reduction of oxidative stress and/or stimulation of cellular degradation systems.
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