Optimization of Cell Viability Assays for Screening Glioprotective Compounds in Primary Rat Optic Nerve Head Astrocytes
Background. Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute to extracellular matrix synthesis. Pathological changes in glaucoma include reactive astrocytosis, a process characterized by altered astrocyte gene and protein expression and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA to slow glaucoma and related pathologies are of high clinical relevance.
Objective. The aim of the paper is the development of standardized methods for the systematic advancement of glioprotective strategies using plate reader-based assays determining cellular viability, plate-reader based proliferation and the intracellular redox state.
Methods. In our work we used primary culture of OHNAs as a model system. The oxidative stress was induced by tBHP. Reactive oxygen species (ROS) were measured by the DCDFA assay. For cell viability tests we used the optimized lactate dehydrogenase (LDH) release assay, as well as the MTT and Calcein-AM uptake assays.
Results. The half-maximal effect (EC50) of tBHP on OHNAs ROS levels in the DCFDA assay was 192.1 ± 15.7 μM. The measurement of cellular viability of OHNAs after tBHP-induced oxidative stress showed EC50 = 156.9 ± 3.8 µM in the Calcein-AM uptake assay. In the MTT assay, the EC50 for tBHP was 138.1 ± 1.4 µM, and shifted to 192.7 ± 2.8 µM with 100 μM Trolox pre-treatment of OHNA. In the LDH release assay, the EC50 for tBHP was 146.9 ± 4.9 µM and 246.3 ± 7.3 µM for the control and Trolox conditions respectively.Conclusions. Our results provide feasibility data for the plate-reader based screening for novel glioprotectants using primary ONHA culture.
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H.A. Quigley, “Glaucoma”, Lancet, vol. 377, no. 9774, pp. 1367–1377, 2011. doi: 10.1016/S0140-6736(10)61423-7
R.N. Weinreb et al., “Primary open-angle glaucoma”, Nat. Rev. Dis. Primers, vol. 2, pp. 16067, 2016. doi: 10.1038/nrdp.2016.67
R.N. Weinreb et al., “The pathophysiology and treatment of glaucoma: A review”, JAMA, vol. 311, no. 18, pp. 1901–1911, 2014. doi: 10.1001/jama.2014.3192
A.J. Payne et al., “Antioxidant drug therapy approaches for neuroprotection in chronic diseases of the retina”, Int. J. Mol. Sci., vol. 15, no. 2, pp. 1865–1886, 2014. doi: 10.3390/ijms15021865
E.E. Chang and J.L. Goldberg, “Glaucoma 2.0: neuroprotection, neuroregeneration, neuroenhancement”, Ophthalmology, vol. 119, no. 5, pp. 979–986, 2012. doi: 10.1016/j.ophtha.2011.11.003
M.R. Hernandez, “The optic nerve head in glaucoma: role of astrocytes in tissue remodeling”, Prog. Retin. Eye Res., vol. 19, no. 3, pp. 297–321, 2000. doi: 10.1016/s1350-9462(99)00017-8
M.R. Hernandez et al., “Astrocytes in glaucomatous optic neuropathy”, Prog. Brain Res., vol. 173, pp. 353–373, 2008. doi: 10.1016/S0079-6123(08)01125-4.
S. Kaja et al., “Differential subcellular Ca(2+) signaling in a highly specialized subpopulation of astrocytes”, Experimental Neurology, vol. 265, pp. 59–68, 2015. doi: 10.1016/j.expneurol.2014.12.014
S. Kaja et al., “An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience”, J. Pharmacol. Toxicol. Methods, vol. 73, pp. 1–6, 2015. doi: 10.1016/j.vascn.2015.02.001
S. Burroughs et al., “Plate reader-based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures”, J. Neurosci. Methods, vol. 203, no. 1, pp. 141–145, 2012. doi: 10.1016/j.jneumeth.2011.09.007
S. Kaja et al., “Novel mechanism of increased Ca2+ release following oxidative stress in neuronal cells involves type 2 inositol-1,4,5-trisphosphate receptors”, Neuroscience, vol. 175, pp. 281–291, 2011. doi: 10.1016/j.neuroscience.2010.11.010
J. Weyermann et al., “A practical note on the use of cytotoxicity assays”, Int. J. Pharm., vol. 288, no. 2, pp. 369–76, 2005. doi: 10.1016/j.ijpharm.2004.09.018
G. Fotakis and J.A. Timbrell, “In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride”, Toxicol. Lett., vol. 160, no. 2, pp. 171–177, 2006. doi: 10.1016/j.toxlet.2005.07.001
H.O. Jauregui et al., “Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes--freshly isolated cells, cell suspensions, and primary monolayer cultures”, In Vitro, vol. 17, no. 12, pp. 1100–1110, 1981. doi: 10.1007/BF02618612
K. Chiba et al., “Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells”, Toxicol. In Vitro, vol. 12, no. 3, pp. 251–258, 1998. doi: 10.1016/S0887-2333(97)00107-0
D. Lobner,“Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?”, J. Neurosci. Methods, vol. 96, no. 2, pp. 147–152, 2000. doi: 10.1016/S0165-0270(99)00193-4
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