The properties of ARPE-19 appear to be changing in ways that may depend on how the cells are maintained and passaged. Caution should be exercised in comparing data between laboratories and in interpreting studies in which only a subset of cells may respond to experimental stimuli. Specialized media promoted the maturation of the adherens junction, but only a partial maturation of the tight junctions.
A culture model has been established to study the gradual development of tight junctions during the embryogenesis of the chick retinal pigment epithelium. This study asks how closely the culture model reflects normal development and how the composition, structure and function of embryonic tight junctions are affected by the apical and basal environments. The study focused on the expression of claudins, the fine-structure of tight junctional strands and the transepithelial electrical resistance. Between embryonic days 7 and 14, patches of junctional strands gradually expanded and coalesced to form a continuous junction, in vivo. Although there was a corresponding increase in claudin expression, different claudins appeared at different times. In culture, the apical and basal environments acted synergistically to promote a continuous network of tight junctions with higher electrical resistance. Independently, pituitary extract or the secretory products of either embryonic fibroblasts or the retina promoted the formation of tight junctions. In combination, three effects were identified. With basally placed fibroblast conditioned medium, apical retinal medium increased transepithelial electrical resistance by affecting structure alone. With basally placed pituitary extract, apical retinal conditioned medium increased transepithelial electrical resistance by affecting structure and by modulating claudin expression in a manner that was consistent with development in vivo. Although embryonic day 7 and 14 cultures in retinal medium exhibited similar structure, the transepithelial electrical resistance of the embryonic day 14 cultures was higher. This higher transepithelial electrical resistance correlated with differences in claudin expression and localization. Therefore, this experimental model can isolate the effects of retinal secretions on structure and claudin expression, and can help us to determine how claudins affect function when structure is held constant.
The results suggest that the death of retinal neurons can be triggered by hypoxia and that P2X(7) activation is involved in the hypoxia-induced death of retinal neurons. P2X(7) antagonists can prevent hypoxia-induced damage in retinal neurons.
PURPOSE.To determine the modification of the glutamate-induced death of retinal neurons by endothelin (ET)-1. METHODS. Cultured retinal neurons from fetal rats were exposed to glutamate (1.0 mM) alone or glutamate with ET-1 (10
Ϫ10-10 Ϫ7 M) for 10 minutes. Neuronal death was assessed by the trypan blue exclusion or TUNEL assays at 2, 6, and 24 hours after the exposure. The effects of adding BQ-123 or BQ-788, ET A , and ET B receptor antagonists, respectively, in combination with ET-1 was also assessed. RESULTS. Immunohistochemical analyses showed that the ETs as well as ET A and ET B receptors were expressed on cultured retinal neurons consisting mainly of amacrine cells. A brief exposure of the cultured retinal neurons to glutamate alone significantly increased the number of dead cells, and the addition of ET-1 with glutamate caused a further significant increase in retinal neuronal death compared with the cells exposed to glutamate alone. A significant increase in neuronal death was detected at doses of 10 nM of ET-1 and higher after a 24-hour exposure (P Ͻ 0.05, Dunnett), whereas brief exposure of neurons to up to 1 M ET-1 alone did not cause delayed cell death of neurons. BQ-123 (10 nM) suppressed the enhancement of retinal toxicity caused by ET-1 (10 nM), whereas BQ-788 had no significant effect. CONCLUSIONS. These results indicate that ET-1 enhances glutamate-induced retinal cell death, possibly through ET A receptors. ET-1 may act synergistically with glutamate to damage retinal neurons under hypoxic conditions. (Invest Ophthalmol Vis Sci. 2005;46:4684 -4690)
ABSTRACT.Purpose: To investigate the effects of high infusion pressure in conjunction with pars plana vitrectomy (PPV) on retinal morphology and function in rabbits. Methods: Pars plana vitrectomy was performed under urethane (0.8 mg ⁄ kg) anaesthesia in the right eye of albino rabbits following phacoemulsification and aspiration (PEA). The left eyes were not touched. After PEA, the animals were divided into two groups. In six eyes, intraocular pressure (IOP) was increased to 80 mmHg for 30 mins (high-pressure group) and in five eyes IOP was maintained at 40 mmHg for 30 mins (low-pressure group). The IOPs were regulated by the height of the bottle of balanced salt solution (BSS) and monitored with a pressure transducer. After the pressure elevation, vitreous fluid was collected to measure the glutamate concentration. Then, PPV was performed for 15 mins in both groups under an infusion pressure of 40 mmHg. In five additional rabbits, PEA alone was performed in the right eye, and vitreous fluid was collected (PEA group). Functional alterations were assessed by recording visual evoked potentials (VEPs) and electroretinograms (ERGs). Ten days after the IOP changes, the animals were killed with intravenous pentobarbital sodium and the eyes were prepared for histological analysis. Damage to retinal ganglion cells (RGCs) was quantified by counting the number of cells in the ganglion cell layer (GCL). The contralateral eyes in the high-pressure group served as controls (n ¼ 6). Results: The mean implicit time (IT) of the VEPs in the high-pressure group was significantly longer than that before the IOP elevation, by 114)124% (p < 0.05, paired t-test), and also than that of control eyes (p < 0.05, ANOVA followed by t-test). No significant changes in the VEPs were detected in either the low-pressure group or the PEA group. There were significantly fewer cells in the GCL in the high-pressure group (24.7 ⁄ mm) than in the control animals (41.4 ⁄ mm; p < 0.05, Dunnett's test). The number of cells in the GCL in the low-pressure and PEA groups did not significantly differ to that in the controls. The amplitudes of the ERG a-and b-waves were not significantly changed (p > 0.05, paired t-test). Conclusions: These results suggest that high infusion pressure in conjunction with PPV leads to morphological and functional changes in the retina. The absence of ERG changes and presence of VEP changes suggest that these changes were due to damage to RGCs, which supports the morphological observations.
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