Viability of primary cultured retinal neurons in a hyperglycemic condition

doi:10.3969/j.issn.1673-5374.2013.05.004

Abstract

Abstract:

The retina of Wistar rats within 1–3 days of birth were dissociated into a retinal cell suspension using 0.05% trypsin digestion. The cell suspension was incubated in Dulbecco’s modified Eagle’s medium for 24 hours, followed by neurobasal medium for 5–7 days. Nissl staining showed that 79.86% of primary cultured retinal cells were positive and immunocytochemical staining showed that the purity of anti-neurofilament heavy chain antibody-positive cells was 71.53%, indicating that the primary culture system of rat retinal neurons was a reliable and stable cell system with neurons as the predominant cell type. The primary cultured retinal neurons were further treated with 0, 5.5, 15, 25, and 35 mM glucose for 24, 48, and 72 hours. The thiazolyl blue tetrazolium bromide test and flow cytometry showed that with increasing glucose concentration and treatment duration, the viability of retinal neurons was reduced, and apoptosis increased. In particular, 35 mM glucose exhibited the most significant effect at 72 hours. Thus, rat retinal neurons treated with 35 mM glucose for 72 hours can be used to simulate a neuronal model of diabetic retinopathy.

Key words: neural regeneration, peripheral nerve injury, retina, neurons, apoptosis, hyperglycemia model, diabetic retinopathy, glucose, grants-supported paper, photographs-containing paper, neuroregeneration

Yu Liu, Xueliang Xu, Renhong Tang, Guoping Chen, Xiang Lei, Limo Gao, Wenjie Li, Yu Chen. Viability of primary cultured retinal neurons in a hyperglycemic condition[J]. Neural Regeneration Research, 2013, 8(5): 410-419.

References

[1] Wu XY, Zhang D, Liu SZ. Primary culture of retinal neurons of newborn rat. Guoji Yanke Zazhi. 2008;8(1):45-46.

[2] Bai HQ, Wang JH, Wang DB, et al. Short period culture of retinal neurons and ganglion cells of neonatal rat in vitro. Yanke Xin Jinzhan. 2004;2(2):107-110.

[3] Yao J, Li XX. In vitro culture of retinal neuron cells of rat. Yanke Yanjiu. 2004;22(4):340-342.

[4] Zhang YJ, Xu HX, Zeng KH, et al. Influence of Taurine on the expression of GFAP and TAUT in the Müller cells from retina in high glucose culture. Xiandai Shengwu Yixue Jinzhan. 2007;7(5):649-652.

[5] Fletcher EL, Phipps JA, Ward MM, et al. Neuronal and glial cell abnormality as predictors of progression of diabetic retinopathy. Curr Pharm Des. 2007;13(26):2699-2712.

[6] Zeng K, Xu H, Mi M, et al. Effects of taurine on glial cells apoptosis and taurine transporter expression in retina under diabetic conditions. Neurochem Res. 2010; 35(10):1566-1574.

[7] McBain VA, Robertson M, Muckersie E, et al. High glucose concentration decreases insulin-like growth factor type 1-mediated mitogen-activated protein kinase activation in bovine retinal endothelial cells. Metabolism. 2003;52(5):547-551.

[8] Kane R, Stevenson L, Godson C, et al. Gremlin gene expression in bovine retinal pericytes exposed to elevated glucose. Br J Ophthalmol. 2005;89(12):1638-1642.

[9] Yuan Z, Feng W, Hong J, et al. p38MAPK and ERK promote nitric oxide production in cultured human retinal pigmented epithelial cells induced by high concentration glucose. Nitric Oxide. 2009;20(1):9-15.

[10] Layton CJ, Wood JP, Chidlow G, et al. Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose-sensitive manner. J Neurochem. 2005;92(3):487-493.

[11] Santiago AR, Cristóvão AJ, Santos PF, et al. High glucose induces caspase-independent cell death in retinal neural cells. Neurobiol Dis. 2007;25(3):464-472.

[12] Takano M, Sango K, Horie H, et al. Diabetes alters neurite regeneration from mouse retinal explants in culture. Neurosci Lett. 1999;275(3):175-178.

[13] Xu HX, Mi MT, Huang GR, et al. Purified retinal ganglion cells cultured in serum-free neurobasal medium. Zhonghua Yandibing Zazhi. 2006;22(3):200-202.

[14] Hattar S, Kumar M, Park A, et al. Central projections of melanopsin-expressing retinal ganglion cells in the mouse. J Comp Neurol. 2006;497(3):326-349.

[15] Liu FL, Niu YJ, Wang JB, et al. Primary culture of retinal neuron cells of neonatal rat. Shandong Daxue Xuebao: Yixueban. 2006;44(7):684-688.

[16] Zheng ZH, Lin L. Theory and Practice of Neurons Culture. Beijing: Science Press. 2002.

[17] Benowitz L, Yin Y. Rewiring the injured CNS: lessons from the optic nerve. Exp Neurol. 2008;209(2):389-398.

[18] Takahashi N, Cummins D, Caprioli J. Rat retinal ganglion cells in cultures. Exp Eye Res. 1991;53(2):565-572.

[19] Carri NG, Rubin K, Gullberg D, et al. Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen. Int J Dev Neurosci. 1992;10(5):393-405.

[20] Seil FJ. The extracellular matrix molecule, laminin, induces purkinje cell dendritic spine proliferation in granule cell depleted cerebellar cultures. Brain Res. 1998; 795(1-2):112-120.

[21] Hayashida Y, Partida GJ, Ishida AT. Dissociation of retinal ganglion cells without enzymes. J Neurosci Methods. 2004;137(1):25-35.

[22] Zhou MH, Zhao LP. The growth effect of tectal extract on neonatal rat retinal neurons cultured on various substrata. Jiepou Xuebao. 1991;22(3):195-198.

[23] Brunetti V, Maiorano G, Rizzello L, et al. Neurons sense nanoscale roughness with nanometer sensitivity. Proc Natl Acad Sci U S A. 2010;107(14):6264-6269.

[24] Wada T, Honda M, Minami I, et al. Highly efficient differentiation and enrichment of spinal motor neurons derived from human and monkey embryonic stem cells. PLoS One. 2009;4(8):e6722.

[25] Graham JM. Isolation of rat and human hippocampal neuron fractions in a discontinuous density gradient. Sci World J. 2002;2:1634-1637.

[26] Lilley S, Robbins J. The rat retinal ganglion cell in culture: an accessible CNS neurone. J Pharmacol Toxicol Methods. 2005;51(3):209-220.

[27] Xu DF, Zhu CT, Liu DM, et al. A comparative study of three sorts of nutrient medium on primary hippocampal neurons culture. Anhui Yike Daxue Xuebao. 2006;41(5):512-514.

[28] Kino T, Jaffe H, Amin ND, et al. Cyclin-dependent kinase 5 modulates the transcriptional activity of the mineralocorticoid receptor and regulates expression of brain-derived neurotrophic factor. Mol Endocrinol. 2010; 24(5):941-952.

[29] Julien JP, Mushynski WE. Neurofilaments in health and disease. Prog Nucleic Acid Res Mol Biol. 1998;61(1):1-23.

[30] Kikuchi M, Kashii S, Honda Y, et al. Protective effects of methylcobalamin, a vitamin B12 analog, against glutamate-induced neurotoxicity in retinal cell culture. Invest Ophthalmol Vis Sci. 1997;38(5):848-854.

[31] Weinberg JB, Chen Y, Jiang N, et al. Inhibition of nitric oxide synthase by cobalamins and cobinamides. Free Radic Biol Med. 2009;46(12):1626-1632.

[32] Xia XG, Yin Z. Protective function of 7-Difluoromethylyl- 5,4’-Di-methoxyl isoflavone on the damage of hyperglycemic simian retinal endothelial cells. Yanke Xin Jinzhan. 2008;28(2);113-115.

[33] Gou L, Moss SE, Alexander RA, et al. Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix. Invest Ophthalmol Vis Sci. 2005;46(1):175-182.

[34] Spalding KL, Rush RA, Harvey AR. Target-derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina. J Neurobiol. 2004; 60(3):319-327.

[35] Hu H, Lu W, Zhang M, et al. Stimulation of the P2X7 receptor kills rat retinal ganglion cells in vivo. Exp Eye Res. 2010;91(3):425-432.

[36] Johnson EC, Guo Y, Cepurna WO, et al. Neurotrophin roles in retinal ganglion cell survival: lessons from rat glaucoma models. Exp Eye Res. 2009;88(4):808-815.

[37] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.