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In vitro organotypic cultivation of adult newt and rat retinas

  • Biology of the Cell
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Abstract

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiGFAP antibody dye demonstrated an increase in the parent Müller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the adult rat retinal spheroid developing in vitro; BrdU-positive cells and multiple mitoses were revealed in this fissue. However, the source of proliferation was not in the peripheral retina, and resident macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt inner retinas.

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References

  • Abrahan, C.E., Insua, M.F., Politi, L.E., et al., Oxidative Stress Promotes Proliferation of Retinal Glia Cells in Vitro, J. Neurosci. Res., 2009, vol. 87, no. 4, pp. 964–977.

    Article  PubMed  CAS  Google Scholar 

  • Ahmad, I., Jang, L., and Pham, H., Identification of Neural Progenitors in the Adult Mammalian Eye, Biochem. Biophys. Res. Commun., 2000, vol. 270, no. 2, pp. 517–521.

    Article  PubMed  CAS  Google Scholar 

  • Alfinito, P.D. and Townes-Anderson, E., Activation of Mislocalized Opsin Kills Rod Cells: A Novel Mechanism for Rod Cell Death in Retinal Disease, Proc. Natl. Acad. Sci. USA, 2002, vol. 99, no. 8, pp. 5655–5660.

    Article  PubMed  CAS  Google Scholar 

  • Bazhin, A.V., Grigoryan, E.N., Tikhomirova, N.K., et al., An Immunohistochemical Study of Localization of the Calcium-Binding Protein Recoverin in Retina of the Newt Pleurodeles Waltl, Izv. Akad. Nauk, Ser. Biol., 2002, vol. 29, no. 4, pp. 427–436.

    Google Scholar 

  • Bringmann, A., Pannicke, T., Grosche, J., et al., Müller Cells in the Healthy and Diseased Retina, Prog. Retin. Eye Res., 2006, vol. 25, no. 4, pp. 397–424.

    Article  PubMed  CAS  Google Scholar 

  • Cameron, D.A., Cellular Proliferation and Neurogenesis in the Injured Retina of Adult Zebrafish, Vis. Neurosci., 2000, vol. 17, no. 5, pp. 789–797.

    Article  PubMed  CAS  Google Scholar 

  • Chang, M.-L., Wu, C.-H., Jiang-Shieh, Y.-F., et al., Reactive Changes of Retinal Astrocytes and Muller Glial Cells in Kainite-Induced Neuroexcitotoxicity, J. Anat., 2007, vol. 210, pp. 54–65.

    Article  PubMed  CAS  Google Scholar 

  • Cicero, S.A., Johnson, D., Reyntjens, S., et al., Cells Previously Identified as Retinal Stem Cells Are Pigmented Ciliary Epithelial Cells, Proc. Natl. Acad. Sci. USA, 2009, vol. 106, no. 16, pp. 6685–6690.

    Article  PubMed  Google Scholar 

  • Engelhardt, M., Bogdahn, U., and Aignera, L., Adult Retinal Pigment Epithelium Cells Express Neural Progenitor Properties and the Neuronal Precursor Protein Doublecortin, Brain Res., 2005, vol. 1040, pp. 98–111.

    Article  PubMed  CAS  Google Scholar 

  • Engelhardt, M., Wachs, F.P., Couillard-Despres, S., and Aigner, L., The Neurogenic Competence of Progenitors from the Postnatal Rat Retina in Vitro, Exp. Eye Res., 2004, vol. 78, no. 5, pp. 1025–1036.

    Article  PubMed  CAS  Google Scholar 

  • Fan, Y. and Bergmann, A., Apoptosis Induced Compensatory Proliferation. The Cell Is Dead. Long Live the Cell!, Trends Cell Biol., 2008, vol. 18, no. 10, pp. 467–473.

    Article  PubMed  CAS  Google Scholar 

  • Fernandez-Bueno, I., Pastor, J.C., Gayoso, M.J., et al., Müller and Macrophage-Like Cell Interactions in an Organotypic Culture of Porcine Neuroretina, Mol. Vision, 2008, vol. 14, pp. 2148–2156.

    CAS  Google Scholar 

  • Fischer, A.J. and Reh, T.A., Identification of a Proliferating Marginal Zone of Retinal Progenitors in Postnatal Chickens, Dev. Biol., 2000, vol. 220, no. 2, pp. 197–210.

    Article  PubMed  CAS  Google Scholar 

  • Fischer, A.J. and Reh, T.A., Müller Glia Is a Potential Source of Neural Regeneration in the Postnatal Chicken Retina, Nat. Neurosci., 2001, vol. 4, pp. 247–252.

    Article  PubMed  CAS  Google Scholar 

  • Fischer, A.J. and Reh, T.A., Potential of Müller Glia to Become Neurogenic Retinal Progenitor Cells, Glia, 2003, vol. 43, no. 1, pp. 70–76.

    Article  PubMed  Google Scholar 

  • Fischer, A.J., Scott, M.A., and Tuten, W., Mitogen-Activated Protein Kinase-Signaling Stimulates Müller Glia to Proliferate in Acutely Damaged Chicken Retina, Glia, 2009, vol. 57, no. 2, pp. 166–181.

    Article  PubMed  Google Scholar 

  • Germer, A., Kuhnel, K., Grosche, J., et al., Development of the Neonatal Rabbit Retina in Organ Culture. 1. Comparison with Histogenesis in Vivo, and Effect of a Gliotoxin (Alfa-Aminoadipic Acid), Anat. Embryol. (Berl.), 1997, vol. 196, no. 1, pp. 67–79.

    Article  CAS  Google Scholar 

  • Grigoryan, E.N., Alternative Intrinsic Cell Sources for Neural Retina Regeneration in Adult Urodelean Amphibians, in Strategies for Retinal Tissue Repair and Regeneration in Vertebrates: From Fish to Human, Chiba, Ch., Ed., India: Trivandrum Res. Singnpost, 2008, pp. 35–62.

    Google Scholar 

  • Grigoryan, E.N. and Poplinskaya, V.A., Discovery of Internal Sources of the Neural Retina Regeneration after Its Detachment in Newts. 2. A Radioautographic Study, Izv. Akad. Nauk, Ser. Biol., 1999, vol. 26, no. 5, pp. 583–591.

    Google Scholar 

  • Grigoryan, E.N., Bazhin, A.V., Krasnov, M.S., and Filippov, P.P., Study of Expression of the Calcium-Binding Protein Recoverin in Normal, Surviving, and Regenerating Retina of Adult Newt Pleurodeles waltl, Klet. Tekhnol. Biol. Med., 2009, no. 3, pp. 173–180.

  • Grigoryan, E.N., Ivanova, I.P., and Poplinskaya, V.A., Discovery of New Internal Sources of the Neural Retina Regeneration after Its Detachment in Newts: Morphological and Quantitative Studies, Izv. Akad. Nauk, Ser. Biol., 1996, no. 3, pp. 319–332.

  • Grigoryan, E.N., Krasnov, M.S., Aleinikova, K.S., et al., Rotational Culturing of Isolated Newt Retina as a Method to Obtain Low-Differentiated Proliferating Cells in Vitro, Dokl. Akad. Nauk, 2005, vol. 404, no. 5, pp. 566–570.

    Google Scholar 

  • Hasegawa, M., Restitution of the Eye After Removal of the Retina and Lens in the Newt Triturus Pyrrhogaster, Embryologia, 1958, vol. 4, no. 1, pp. 1–32.

    Article  Google Scholar 

  • Johnson, T.V. and Martin, K.R., Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an in Vitro Intraocular Stem Cell Transplantation Model, Invest. Ophthalmol. Vis. Sci., 2008, vol. 49, no. 8, pp. 3503–3512.

    Article  PubMed  Google Scholar 

  • Kaempf, S., Walter, P., Salz, A.K., and Thumann, G., Novel Organotypic Culture Model of Adult Mammalian Neurosensory Retina in Co-Culture with Retinal Pigment Epithelium, J. Neurosci. Methods, 2008, vol. 173, no. 1, pp. 47–58.

    Article  PubMed  Google Scholar 

  • Keefe, J.R., An Analysis of Urodelean Retinal Regeneration, J. Exp. Zool., 1973, vol. 184, pp. 185–257.

    Article  PubMed  CAS  Google Scholar 

  • Koizumi, A., Zeck, G., Ben, Y., et al., Organotypic Culture of Physiologically Functional Adult Mammalian Retinas, PLoS ONE, 2007, vol. 2, no. 2, p. e221.

    Article  PubMed  CAS  Google Scholar 

  • Kustermann, S., Schmid, S., Biehlmaier, O., and Kohler, K., Survival, Excitability, and Transfection of Retinal Neurons in an Organotypic Culture of Mature Zebrafish Retina, Cell Tissue Res., 2008, vol. 332, no. 2, pp. 195–209.

    Article  PubMed  Google Scholar 

  • Lewis, G.P. and Fisher, S.K., Up-Regulation of Glial Fibrillary Acidic Protein in Response to Retinal Injury: Its Potential Role in Glial Remodeling and a Comparison to Vimentin Expression, Int. Rev. Cytol., 2003, vol. 230, pp. 263–290.

    Article  PubMed  CAS  Google Scholar 

  • Liljekvist-Larsson, I., Torngren, M., Abrahamson, M., and Johansson, K., Growth of the Postnatal Rat Retina in Vitro: Quantitative RT-PCR Analyses of MRNA Expression for Photoreceptor Proteins, Mol. Vision, 2003, vol. 9, pp. 657–664.

    CAS  Google Scholar 

  • Mitashov, V.I. and Maliovanova, S.D., Cellular Proliferative Potentials of the Pigment and Ciliated Epithelium of the Eye in Clawed Toads Normally and during Regeneration, Ontogenez, 1982, vol. 13, no. 3, pp. 228–234.

    PubMed  CAS  Google Scholar 

  • Mitashov, V.I., Arsanto, J.P., Markitantova, Y.V., and Thouveny, Y., Remodeling Processes during Neural Retinal Regeneration in Adult Urodeles: An Immunohistochemical Survey, Int. J. Dev. Biol., 1995, vol. 39, no. 6, pp. 993–1003.

    PubMed  CAS  Google Scholar 

  • Mitashov, V.I., Autoradiographic Study of Retina Regeneration in Crested Newts Triturus sristatus, Dokl. Akad. Nauk SSSR, 1968, vol. 181, no. 6, pp. 1510–1513.

    PubMed  CAS  Google Scholar 

  • Mitashov, V.I., Retinal Regeneration in Amphibians, Int. J. Dev. Biol., 1997, vol. 41, pp. 893–905.

    PubMed  CAS  Google Scholar 

  • Monnin, J., Morand-Villeneuve, N., Michel, G., et al., Production of Neurospheres from Mammalian Müller Cells in Culture, Neurosci. Lett., 2007, vol. 421, no. 1, pp. 22–26.

    Article  PubMed  CAS  Google Scholar 

  • Ng, T.F., Kitaichi, N., and Taylor, A.W., In Vitro Generated Autoimmune Regulatory T Cells Enhance Intravitreous Allogenic Retinal Graft Survival, Invest. Ophthalmol. Vis. Sci., 2007, vol. 48, no. 11, pp. 5112–5117.

    Article  PubMed  Google Scholar 

  • Novikova, Yu.P., Poplinskaya, V.A., Aleinikova, K.S., and Grigoryan, E.N., A Study of the Localization and Accumulation of S-Phase Cells in the Retina of Newt Pleurodeles waltl after Experimental Pigment Epithelial Detachment) Ontogenez, 2008, vol. 39, no. 2, pp. 143–150.

    PubMed  Google Scholar 

  • Ooto, S., Akagi, T., Kageyama, R., et al., Potential for Neural Regeneration after Neurotoxic Injury in the Adult Mammalian Retina, Proc. Natl. Acad. Sci. USA, 2004, vol. 101, no. 37, pp. 13654–13659.

    Article  PubMed  CAS  Google Scholar 

  • Perron, M. and Harris, W.A., Retinal Stem Cells in Vertebrates, BioEssays, 2000, vol. 22, no. 8, pp. 685–688.

    Article  PubMed  CAS  Google Scholar 

  • Poplinskaya, V.A., Cytostructure and Morphogenesis of Rod Segments, Ontogenez, 1995, vol. 26, no. 1, pp. 5–21.

    Google Scholar 

  • Raad, DeS., Comte, M., Nef, P., et al., Distribution Pattern of Three Neural Calcium-Binding Proteins (NSC-1, VILIP and Recoverin) in Chicken, Bovine and Rat Retina, Histochem. J., 1995, vol. 27, pp. 524–535.

    PubMed  Google Scholar 

  • Raymond, P.A. and Hitchcock, P.F., Retinal Regeneration: Common Principles but a Diversity of Mechanisms, Adv. Neurol., 1997, vol. 72, pp. 171–184.

    PubMed  CAS  Google Scholar 

  • Raymond, P.A., Barthel, L.K., Bernardos, R.L., and Perkowski, J.J., Molecular Characterization of Retinal Stem Cells and Their Niches in Adult Zebrafish, BMC Dev. Biol, 2006, vol. 6, p. 36.

    Article  PubMed  CAS  Google Scholar 

  • Raymond, P.A., Retinal Regeneration in Teleost Fish, Ciba Found. Symp., 1991, vol. 160, pp. 171–186.

    PubMed  CAS  Google Scholar 

  • Reh, T.A. and Fischer, A.J., Retinal Stem Cells, Methods Enzymol., 2006, vol. 419, pp. 52–73.

    Article  PubMed  CAS  Google Scholar 

  • Romano, C. and Hicks, D., Adult Retinal Neuronal Cell Culture, Prog. Ret. Eye Res., 2007, vol. 26, pp. 379–397.

    Article  CAS  Google Scholar 

  • Stepanik, P.L., Lerious, V., and McGinnis, J.F., Developmental Appearance, Species and Tissue Specificity of Mouse 23-kDa, a Retinal Calcium-Binding Protein (Recoverin), Exp. Eye Res., 1993, vol. 57, no. 2, pp. 189–197.

    Article  PubMed  CAS  Google Scholar 

  • Sullivan, R., Penfold, P., and Pow, D.V., Neuronal Migration and Glial Remodeling in Degenerating Retinas of Aged Rats and in Nonneovascular AMD, Invest. Ophthalmol. Vis. Sci., 2003, vol. 44, pp. 856–865.

    Article  PubMed  Google Scholar 

  • Tropepe, V., Retinal Stem Cells in the Adult Mouse Eye, Science, 2000, vol. 287, pp. 2032–2036.

    Article  PubMed  CAS  Google Scholar 

  • Vihtelic, T.S. and Hyde, D.R., Light-Induced Rod and Cone Cell Death and Regeneration in the Adult Albino Zebrafish (Danio rerio) Retina, J. Neurobiol., 2000, vol. 44, no. 3, pp. 289–307.

    Article  PubMed  CAS  Google Scholar 

  • Viktorov, I.V., Aleksandrova, O.P., and Alekseeva, N.Y., Roller Organ Culture of the Retina from Postnatal Rats, Bull. Exp. Biol. Med., 2006, vol. 142, no. 4, pp. 486–489.

    Article  PubMed  CAS  Google Scholar 

  • Wan, J., Zheng, H., Chen, Z.L., et al., Preferential Regeneration of Photoreceptor from Müller Glia after Retinal Degeneration in Adult Rat, Vision Res., 2008, vol. 48, no. 2, pp. 223–234.

    Article  PubMed  CAS  Google Scholar 

  • Willbold, E., Berger, J., Reinicke, M., and Wolburg, H., On the Role of Müller Glia Cells in Histogenesis: Only Retinal Spheroids, but Not Tectal, Telencephalic and Cerebellar Spheroids Develop Histotypical Patterns, J. Hirnforsch., 1997, vol. 38, no. 3, pp. 383–396.

    PubMed  CAS  Google Scholar 

  • Woodford, B.J. and Blanks, J.C., Uptake of Tritiated Thymidine in Mitochondria of the Retina, Invest. Ophthalmol. Vis. Sci., 1989, vol. 30, no. 12, pp. 2528–2532.

    PubMed  CAS  Google Scholar 

  • Xue, L.P., Lu, J., Hu, S., et al., Muller Glial Cells Express Nestin Coupled with Glial Fibrillary Acidic Protein in Experimentally Induced Glaucoma in the Rat Retina, Neuroscience, 2006, vol. 139, no. 2, pp. 723–732.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Yu. P. Novikova.

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Original Russian Text © Yu.P. Novikova, K.S. Aleynikova, M.S. Krasnov, V.A. Poplinskaya, E.N. Grygoryan, 2010, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2010, No. 4, pp. 389–402.

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Novikova, Y.P., Aleynikova, K.S., Krasnov, M.S. et al. In vitro organotypic cultivation of adult newt and rat retinas. Biol Bull Russ Acad Sci 37, 327–338 (2010). https://doi.org/10.1134/S1062359010040011

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