Time course modifications in organotypic culture of human neuroretina

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Títol: Time course modifications in organotypic culture of human neuroretina
Autors: Fernández Bueno, Iván | Fernández-Sánchez, Laura | Gayoso Rodríguez, Manuel J. | García Gutiérrez, María T. | Pastor, José C. | Cuenca, Nicolás
Grups d'investigació o GITE: Neurobiología del Sistema Visual y Terapia de Enfermedades Neurodegenerativas (NEUROVIS)
Centre, Departament o Servei: Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología
Paraules clau: Human retina | Organotypic culture | Photoreceptors | Bipolar cells | Horizontal cells | Glial cells | Neurodegeneration
Àrees de coneixement: Biología Celular
Data de publicació: 26-de setembre-2012
Editor: Elsevier
Citació bibliogràfica: FERNANDEZ-BUENO, Iván, et al. "Time course modifications in organotypic culture of human neuroretina". Experimental Eye Research. Vol. 104 (Nov. 2012). ISSN 0014-4835, pp. 26-38
Resum: The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell® dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.
Patrocinadors: This research was supported by grants from the Spanish Ministry (BFU2009-07793/BFI and SAF2007-62262), Instituto de Salud Carlos III (RETICS RD07/0062/0012), ONCE, Fundaluce and Fundación Médica Mutua Madrileña to NC; and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León (Spain) to JCP.
URI: http://hdl.handle.net/10045/25070
ISSN: 0014-4835 (Print) | 1096-0007 (Online)
DOI: 10.1016/j.exer.2012.08.012
Idioma: eng
Tipus: info:eu-repo/semantics/article
Revisió científica: si
Versió de l'editor: http://dx.doi.org/10.1016/j.exer.2012.08.012
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