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John P M Wood, Glyn Chidlow, Robert James Casson; Relative susceptibility of individual neuron classes to different insults in rat retinal cultures. Invest. Ophthalmol. Vis. Sci. 2016;57(12):753. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the relative susceptibility of different classes of cultured retinal neuron to distinct insults: metabolic compromise, excitotoxicity and oxidative stress.
Retinal cultures were prepared from neonatal rat pups via a mechanical and enzymatic digest; cultures were allowed to grow for 7 days before treatment and each treatment lasted 16 hours. Neurons were classified according to immunolabelling for combinations of discrete markers (GABA, tau, βIII-tubulin, MAP2, calretinin, syntaxin, PGP9.5, synaptophysin, NeuN). Cells were treated with sodium azide (NaN3; 500µM) to simulate metabolic compromise, kainic acid (KA; 50µM) to induce excitotoxicity or t-butyl hydroperoxide (t-bH; 1µM) to bring about oxidative stress. After treatments, surviving cells were quantified by immunocytochemistry, Western blot or the absence of apoptosis (TUNEL); cell loss for analysed markers was quantified in each case either by cell/protein quantification or by using ImageJ, where appropriate.
All neurons in the cultures (except for photoreceptors) labelled positively for tau; 35±5% of tau-positive neurons remained after treatment with KA, 53±7% after treatment with t-bH and 19±4% after treatment with NaN3. Different subsets of the tau-positive neurons labelled with combinations of GABA, βIII-tubulin, MAP2, syntaxin, PGP9.5, synaptophysin or NeuN; interestingly, tau/calretinin neurons did not label with any other marker. GABA/tau- and calretinin/tau-labelled neurons were the most susceptible to KA, being reduced to 5±3% and 13±5% of control values respectively; syntaxin/tau neurons were the least susceptible, with 43±11% remaining after insult. PGP9.5/tau neurons were the most affected by NaN3 with just 8±5% remaining after treatment; syntaxin/tau neurons were the least affected (72±7% remaining). Calretinin/tau neurons were the most susceptible to t-bH (14±6% remaining after treatment); βIII-tubulin/tau neurons were the least susceptible (68±8% remaining).
Cultured retinal neurons display differential susceptibilities to both specific (excitotoxic, KA) and to more general (oxidative stress, t-bH; metabolic compromise, NaN3) toxic agents. Translation of the present data to the in vivo situation may allow predictions of specific retinal neuron class susceptibility to specific injurious situations and diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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