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|Title:||Mitochondrial inhibition in rat retinal cell cultures as a model of metabolic compromise: mechanisms of injury and neuroprotection|
|Citation:||Investigative Ophthalmology & Visual Science, 2012; 53(8):4897-4909|
|Publisher:||Assoc Research Vision Ophthalmology Inc|
|John P. M. Wood, Teresa Mammone, Glyn Chidlow, Tim Greenwell, and Robert J. Casson|
|Abstract:||PURPOSE. Our study aimed to establish a model of energetic and metabolic dysfunction to cultured retinal cells by chemically inhibiting the mitochondrial electron transport chain with sodium azide (NaN₃), and subsequently investigating toxic mechanisms and potential neuroprotective strategies. METHODS. Mixed rat retinal cultures comprising neurons and glia were treated with a range of NaN₃ concentrations for up to 24 hours and toxicity levels were determined by immunologic methods. Detailed pathologic mechanisms were investigated by assessing apoptosis (TUNEL assay), mitochondrial membrane potential, reactive oxygen species (ROS), and levels of adenosine triphosphate (ATP). Finally, a number of pharmacologic agents were tested to determine whether they could abrogate the effects of NaN₃ to retinal cells. RESULTS. Neurons and glia were killed by NaN3 in a concentration- and time-dependent manner, with neurons being relatively more susceptible. Cell loss was via apoptosis for glia but not for neurons. Cell death generally involved a loss of mitochondrial membrane potential, a reduction in cellular ATP, and an increase in intracellular ROS levels. Glucose was partially able to prevent neuron death, as were the antioxidants trolox and pyruvate, calpain inhibitor III, the ryanodine receptor blocker dantrolene, and the nitric oxide synthase inhibitor L-NAME. CONCLUSIONS. Mitochondrial respiratory inhibition via NaN3 treatment, with delineated mechanisms of toxicity and neuroprotection, represents a valid and reproducible metabolic challenge to cultured retinal cells.|
|Keywords:||Neuroglia; Retinal Ganglion Cells; Retina; Cells, Cultured; Mitochondria; Animals; Rats; Rats, Sprague-Dawley; Mitochondrial Diseases; Sodium Azide; Reactive Oxygen Species; Glucose; Adenosine Triphosphate; Neuroprotective Agents; Enzyme Inhibitors; Antioxidants; Blotting, Western; Immunohistochemistry; Apoptosis; Membrane Potentials|
|Rights:||Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc.|
|Appears in Collections:||Opthalmology & Visual Sciences publications|
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