Journal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
Volume 58, Issue 7, Pages 2915-2921Publisher
ASSOC RESEARCH VISION OPHTHALMOLOGY INC
DOI: 10.1167/iovs.16-21355
Keywords
high glucose; diabetes; diabetic retinopathy; retinal Muller cells; mitochondria
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Funding
- National Institutes of Health [EY018218, EY025528]
- Undergraduate Research Opportunities Program award at Boston University
- Medical Student Summer Research Program award at Boston University School of Medicine
- Research to Prevent Blindness, Inc.
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PURPOSE. To investigate whether high glucose (HG) induces mitochondrial dysfunction and promotes apoptosis in retinal Muller cells. METHODS. Rat retinal Muller cells (rMC-1) grown in normal (N) or HG (30 mM glucose) medium for 7 days were subjected to MitoTracker Red staining to identify the mitochondrial network. Digital images of mitochondria were captured in live cells under confocal microscopy and analyzed for mitochondrial morphology changes based on form factor (FF) and aspect ratio (AR) values. Mitochondrial metabolic function was assessed by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a bioenergetic analyzer. Cells undergoing apoptosis were identified by differential dye staining and TUNEL assay, and cytochrome c levels were assessed by Western blot analysis. RESULTS. Cells grown in HG exhibited significantly increased mitochondrial fragmentation compared to those grown in N medium (FF = 1.7 +/- 0.1 vs. 2.3 +/- 0.1; AR = 2.1 +/- 0.1 vs. 2.5 +/- 0.2; P < 0.01). OCR and ECAR were significantly reduced in cells grown in HG medium compared to those grown in N medium (steady state: 75% +/- 20% of control, P < 0.02; 64% +/- 22% of control, P < 0.02, respectively). These cells also exhibited a significant increase (similar to 2-fold) in the number of apoptotic cells compared to those grown in N medium (P < 0.01), with a concomitant increase in cytochrome c levels (247% +/- 94% of control, P < 0.05). CONCLUSIONS. Findings indicate that HG-induced mitochondrial morphology changes and subsequent mitochondrial dysfunction may contribute to retinal Muller cell loss associated with diabetic retinopathy.
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