April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Chemically-induced Anoxia in Retinal Cell Cultures: Mechanisms of Injury and Neuroprotection
Author Affiliations & Notes
  • John P. Wood
    Ophthalmic Research Laboratories, S Australian Institute of Ophthalmology, Adelaide, Australia
  • Glyn Chidlow
    Ophthalmic Research Laboratories, S Australian Institute of Ophthalmology, Adelaide, Australia
  • Teresa Mammone
    Ophthalmic Research Laboratories, S Australian Institute of Ophthalmology, Adelaide, Australia
  • Robert J. Casson
    Ophthalmic Research Laboratories, S Australian Institute of Ophthalmology, Adelaide, Australia
  • Footnotes
    Commercial Relationships  John P. Wood, None; Glyn Chidlow, None; Teresa Mammone, None; Robert J. Casson, None
  • Footnotes
    Support  NHMRC grant number 565202
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4445. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      John P. Wood, Glyn Chidlow, Teresa Mammone, Robert J. Casson; Chemically-induced Anoxia in Retinal Cell Cultures: Mechanisms of Injury and Neuroprotection. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4445.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: : To use the mitochondrial inhibitor, sodium azide (NaN3), which blocks electron transfer between cytochrome C oxidase and oxygen, to induce a reproducible injury to cultured retinal cells by chemically mimicking anoxia. Furthermore, to determine and attempt to prevent the pathological intracellular processes which result from this insult.

Methods: : Retinal cultures were prepared from 2 day old Sprague-Dawley rat pups via a mechanical and enzymatic digestion procedure. After 7 days, cultures were treated with NaN3 (range of concentrations from 1µM up to 100mM) for 24 hours. Toxicological effects to retinal neurons and glia were delineated by immunocytochemistry, immunoblotting, TUNEL assay and cell viability determinations. Attempts were made to abrogate the toxic effects of NaN3 by using a range of inhibitory agents.

Results: : Retinal cells in culture were dose-dependently killed by NaN3, with neurons being much more sensitive to the treatment than glia (Muller cells, astrocytes and microglia), as assessed by GABA- tau-, PGP9.5-, βIII-tubulin-, nestin-, vimentin-, GFAP- and ED-1-immunolabelling and confirmed by immunoblotting and viability assays. After a 24 hour incubation, 50% of neurons were killed by 245µM and 50% of glia by 10mM NaN3. Death of neurons but not glia was via apoptosis as assessed by TUNEL assay. The determined neuronal death could be partially blocked by the antioxidant, trolox (10µM), the NOS inhibitor, L-NAME (10µM), sodium pyruvate (5mM), the ryanodine receptor blocker, dantrolene (10µM) and by the calpain inhibitor, MDL38170 (1µM).

Conclusions: : Treatment with NaN3 proved to provide a reproducible means to mimic an anoxic insult to retinal cells, and particularly neurons, in culture. Co-incubation with specific classes of inhibitory compounds delineated that neuron death occurred through an apoptotic process involving free radical production, release of calcium from intracellular stores and calpain activation. These data have implications for the treatment of energetically-compromised retinal neurons.

Keywords: retinal culture • apoptosis/cell death • hypoxia 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×