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(Investigative Ophthalmology and Visual Science. 2003;44:818-825.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.01-1144
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Partial Characterization of Retina-Derived Cone Neuroprotection in Two Culture Models of Photoreceptor Degeneration

Anne-Claire Fintz,1,2,3 Isabelle Audo,1,2,4 David Hicks,1 Saddek Mohand-Said,1 Thierry Léveillard,1 and José Sahel1

1From the Laboratory of Molecular and Cellular Pathophysiology of the Retina, National Institute of Health and Medical Research (INSERM), Strasbourg, France.

PURPOSE. To define the nature and estimate the molecular weight range of soluble endogenous retinal trophic activities on cone photoreceptor survival in two models of cone degeneration.

METHODS. Diffusible factors from dissociated retinal cell cultures of 8-day normal-sighted (C57BL/6J) mice were tested for cone-survival-promoting activity by two approaches and by using two independent photoreceptor degeneration models. In the first approach, mouse retinal cells were cultured on semi-permeable membranes apposed to dissociated cultures of chick embryo retina. In the second approach, conditioned medium was collected from normal mouse retinal cultures and added to embryonic chicken retina cultures or to retinal explants obtained from 5-week retinal degeneration (rd1) mice. In some experiments, conditioned medium was heated or sequentially fractionated in dialysis tubing with molecular weight cutoffs of 8, 15, and 25 kDa. The number of chicken cones and viability were determined by using morphologic criteria, colorimetric assays, and labeling with antibodies raised against visinin. Mouse cones were counted by differential double immunolabeling with antibodies against rhodopsin (rods) and arrestin (rods and cones).

RESULTS. Coculturing with normal mouse retinal cells delayed cone loss in dispersed embryonic chicken retina, by a maximum of 50% relative to the control. Conditioned medium derived from normal mouse retinas also significantly delayed cone loss in chicken cone cultures by a maximum of 1300%, compared with the control, and 40% in rd1 mouse retinal explant cultures. The survival activity in conditioned medium was destroyed by heat denaturation, and was partially retained by dialysis with a molecular weight cutoff of 25 kDa in both models.

CONCLUSIONS. These strategies have identified cone-survival-stimulating activities in normal mouse retina, capable of acting across species and enhancing both structural protection and viability. Such molecules may represent candidates for clinical treatment of inherited retinal degeneration.




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