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Jose-Carlos Pastor, Ivan Fernandez-Bueno, Laura Fernandez-Sanchez, Maria Teresa Garcia-Gutierrez, Manuel Gayoso, Nicolas Cuenca; Time Course Of Organotypic Culture Degeneration Of Human Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5366.
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To characterise an ex-vivo adult human retina organ culture, to establish the survival pattern and early degeneration of neural and glial cells
Two postmortem human globes from donors aged 57 and 59 years old were dissected and 16 neuroretina explants (5x5 mm) were obtained. Four were used as freshly retina controls and 12 were explanted on Transwell® culture dishes. Culture medium composed of Neurobasal A supplemented with B-27, FBS and L-glutamine was used. Cultured explants were kept for 3 (n=4), 6 (n=4) and 9 (n=4) days. Vertical epoxi resin sections, stained with toluidine blue, and cryostat sections, singly or doubly immunostained for specific markers of neuronal and glial cells, were used for evaluation.
Retinal morphology was properly preserved at culture start-point. Progressive retinal degeneration was observed, including early pyknosis of photoreceptor nuclei; cellular vacuolization in the ganglion cell layer; diminution of the plexiform layers thicknes; disruption and truncation of photoreceptor outer segments (OS); and marked reduction in the number of nuclei at both nuclear layers, where cells appeared less densely packed. Remarkable changes at 3 days included swelling of cone OS with impaired of their pedicles; loss of axon and dendrites of horizontal and rod bipolar cells stained with antibodies against calbindin (CB) and protein kinase C (PKC), respectively. At end-point (9 days), horizontal cells presented pyknotic nuclei and their terminal tips were gone. Similar degenerative processes were found in the outer plexiform layer for rod bipolar cells and a loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal an increase of gliosis by Müller cells. However, some Müller cells were CB inmunoreactive since 6 days of culture
These morphological changes, early observed at culture in cones, particularly in their OS and pedicles, and in their postsynaptic cells (horizontal and bipolar cells), no previously describe in human retina samples, indicate the important relationship of the retina with the pigment epithelium. These results may suggest the importance of time in retinal detachment treatment
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