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      Development of the Embryonic Porcine Neuroretina in vitro

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          Purpose: The objective of this study was to investigate the survival and morphology of embryonic porcine full-thickness neuroretina in culture. Methods: Porcine fetuses were taken out by cesarian section, and the eyes were enucleated. Neuroretinas were explanted on culture plate inserts and were kept for 0–42 days in vitro under standard culture conditions. Green nucleic acid (Sytox) was used for measuring the extent of cell death, and 4,6-diaminidine-2-phenylindoldihydrochloride was used as a marker for the cellular layers. The explants were examined as whole-mount preparations and vertical sections. Sectioned tissue was stained with hematoxylin-eosin and labeled for immunohistochemistry with photoreceptor-specific antibodies raised against transducin and recoverin. Results: In explants kept for 0–5 days in vitro, the developing retina consisted of multiple rows of neuroblastic cells and a more defined, but multilayered ganglion cell layer (GCL). Older explants revealed a more differentiated appearance with ultimately all normal retinal layers present, even after 42 days in vitro. Transducin- and recoverin-labeled photoreceptors were seen in these specimens, but no outer segments were found. The whole-mount preparation revealed extensively Sytox-labeled cells in the GCL at 2 days in vitro, but very few cells were labeled in older explants. Conclusion: This study shows that cultured fetal porcine full-thickness neuroretina can survive and develop according to its intrinsic timetable for at least 6 weeks in vitro. The in vitro system for culturing of the full-thickness retina may be useful in experiments involving retinal transplantation.

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          Most cited references 17

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          Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats.

          To investigate the short- and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent marker diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an initial abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons.
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            Basic fibroblast and epidermal growth factors stimulate survival in adult porcine photoreceptor cell cultures.

            To investigate the effects of basic fibroblast and epidermal growth factor (FGF2 and EGF, respectively) on the survival and phenotypic expression of photoreceptors isolated from adult mammalian retinas. Primary cultures highly enriched in photoreceptors were prepared from adult domestic pig retinas and maintained in chemically defined medium. Cell culture composition was characterized through the use of specific antibody markers of retinal neurons, and neuronal survival was quantified through viability assays as a function of time in the presence or absence of different doses of FGF2 and EGF. Western blot analysis of phosphotyrosine residues was used to monitor activation of FGF2 and EGF signaling pathways. Reproducible survival of adult pig rod and cone photoreceptors was obtained for approximately 2 weeks in vitro, with the continued expression of rod opsin, recoverin, S-antigen, cone arrestin, and neuron-specific enolase. Purity of cultures was routinely more than 95% photoreceptors, with a rod-cone ratio of 2:3.1. Photoreceptor survival was stable for the initial week, decreasing slowly during the second, with rapid cell loss occurring thereafter. In the presence of FGF2 (20 ng/mL), the percentage of photoreceptor survival during the second week in culture was statistically significantly different, at least two times higher than in control experiments. Photoreceptor survival correlated directly with increasing concentrations of FGF2, and also of EGF. Combined treatment with FGF2 and EGF did not induce higher survival than either factor alone. There was no detectable selective loss of rods or cones in the experimental model. Phosphotyrosine immunoblots after stimulation of cultures with FGF2 and EGF revealed time-dependent appearance of multiple immunoreactive bands. The adult pig photoreceptor culture in the current study exhibits reproducible neuronal survival in vitro and represents a useful novel experimental system for the study of potential neuroprotective effects and signaling pathways of neurotrophic factors such as FGF2 and EGF in fully adult higher mammalian retina.
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              Apoptosis in adult retinal ganglion cells after axotomy.

              Lesions to the mature mammalian central nervous system cause irreversible degeneration, in which neurons have been previously thought to be passive victims. In this study, axon-lesioned adult rat neurons are shown instead to actively degrade themselves through the process of apoptosis: a programmed type of cell death in which the cellular apparatus is actively involved in the degradation process. To investigate whether retinal ganglion cells of an adult mammal follow an apoptotic type of death when their axons are severed, DNA breaks in nuclei were labeled in situ, using a method that specifically incorporates biotinylated deoxynucleotides by exogenous terminal deoxynucleotidyl transferase on the 3'-OH ends of DNA. The active nature of the death mechanism was demonstrated by the reduction in biotin-labeled nuclei after administering the protein synthesis inhibitor cycloheximide. Our results suggest that retinal ganglion cells of the adult rat die through apoptosis when axotomized. This raises new possibilities in the treatment of CNS injuries, by the potential interruptibility of a program for neuronal death.

                Author and article information

                Ophthalmic Res
                Ophthalmic Research
                S. Karger AG
                April 2005
                20 May 2005
                : 37
                : 2
                : 104-111
                Department of Ophthalmology, University Hospital, Lund, Sweden
                84252 Ophthalmic Res 2005;37:104–111
                © 2005 S. Karger AG, Basel

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                Page count
                Figures: 2, References: 27, Pages: 8
                Original Paper


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