Changes in ocular aquaporin expression following optic nerve crush.

PURPOSE: Changes in the expression of water channels (aquaporins; AQP) have been reported in several diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush (ONC). This study was designed to analyze changes in the expression of AQP4 (water selective channel) and AQP9 (water and lactate channel) following ONC in the rat. METHODS: Rat retinal ganglion cells (RGCs) were retrogradely labeled by applying FluoroGold onto the left superior colliculus 1 week before ONC. Retinal injuries were induced by ONC unilaterally. Real-time PCR was used to measure changes in AQP4, AQP9, thy-1, Kir4.1 (K(+) channel), and beta-actin messages. Changes in AQP4, AQP9, Kir4.1, B cell lymphoma-x (bcl-xl), and glial fibrillary acidic protein (GFAP) expression were measured in total retinal extracts using western blotting. RESULTS: The number of RGCs labeled retrogradely from the superior colliculus was 2,090+/-85 cells/mm(2) in rats without any treatment, which decreased to 1,091+/-78 (47% loss) and 497+/-87 cells/mm(2) (76% loss) on days 7 and 14, respectively. AQP4, Kir4.1, and thy-1 protein levels decreased at days 2, 7, and 14, which paralleled a similar reduction in mRNA levels, with the exception of Kir4.1 mRNA at day 2 showing an apparent upregulation. In contrast, AQP9 mRNA and protein levels showed opposite changes to those observed for the latter targets. Whereas AQP9 mRNA increased at days 2 and 14, protein levels decreased at both time points. AQP9 mRNA decreased at day 7, while protein levels increased. GFAP (a marker of astrogliosis) remained upregulated at days 2, 7, and 14, while bcl-xl (anti-apoptotic) decreased. CONCLUSIONS: The reduced expression of AQP4 and Kir4.1 suggests dysfunctional ion coupling in retina following ONC and likely impaired retinal function. The sustained increase in GFAP indicates astrogliosis, while the decreased bcl-xl protein level suggests a commitment to cellular death, as clearly shown by the reduction in the RGC population and decreased thy-1 expression. Changes in AQP9 expression suggest a contribution of the channel to retinal ganglion cell death and response of distinct amacrine cells known to express AQP9 following traumatic injuries.

Involvement of P2X7 receptors in the hypoxia-induced death of rat retinal neurons.

PURPOSE: To investigate the hypoxia-induced death of rat retinal neurons and to determine whether P2X(7) activation is involved in this type of neuronal death. METHODS: Cultured retinal neurons from fetal rats were used. The effects and time course of various degrees of hypoxia (1%-5% O(2)) in the death of retinal neurons, were examined. The effects of P2X(7) antagonists, oxidized adenosine triphosphate (oxidized ATP; 30-100 microM), and brilliant blue G (BBG; 100 nM-10 microM) on hypoxia-induced neuronal death, including apoptosis, were assessed by using trypan blue exclusion, TUNEL assays, and cleaved caspase-3 immunoreactivity. Immunocytochemical analysis was performed to determine whether these neurons express P2X(7) receptors. The effects of P2X(7) receptor stimulation, induced by the P2X(7) agonist benzoyl- benzoyl-ATP (BzATP), on neuronal viability and intracellular Ca(2+) levels ([Ca(2+)](i)) were examined. RESULTS: Retinal neuronal death increased according to the degree of hypoxia and became more severe after 12 hours. Both oxidized ATP and BBG significantly decreased hypoxia-induced neuronal death. Immunocytochemistry demonstrated that P2X(7) receptors were expressed by the cultured retinal neurons. ATP and BzATP caused P2X(7) receptor-dependent neuronal death in a dose-dependent manner and led to a sustained increase in [Ca(2+)](i), with BzATP being more effective than ATP. These effects were hypoxia-induced factor-1alpha- independent and were prevented by oxidized ATP. CONCLUSIONS: 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.

Endothelin-1 (ET-1) is increased in rat retina after crushing optic nerve.

PURPOSE: To determine the alterations in the expression of endothelin-1 (ET-1) and its specific receptors in the sensory retina after optic nerve injury. METHODS: The optic nerve of the right eye of Wistar rats was crushed. The sensory retinas were removed 1, 2, 4, 7, and 14 days after the surgery, and the amount of ET-1 in the retinas was measured by radioimmunoassay, and the mRNA levels of ET(A) and ET(B) receptors were determined by real-time PCR. RESULTS: The ET-1 levels in the sensory retinas following the optic nerve crush were 8.83 +/- 2.23 and 9.99 +/- 4.83 pg/mg protein on days 7 and 14 after the injury, while those in the sham controls were 4.55 +/- 1.36 and 4.85 +/- 1.57 pg/mg protein, respectively. The increase was significant on day 7 (p = 0.003, ANOVA followed by t- test), but not on day 14 (p = 0.054). The mRNA levels of the ET(A) and ET(B) receptors in the retina after the optic nerve crush was significantly increased on day 7. A two-fold increase was recognized in ET(B) receptors. Immunohistochemical analyses revealed that the ET-1 expression was increased mainly in the inner retinal layers, including the nerve fiber layer and ganglion cell layer (GCL). The increased immunoreactivity of the ET(B) receptor was seen in the GCL cells and also in the outer nuclear layer. CONCLUSION: An increase in the intraretinal ET-1 may be involved in the retinal remodeling after the optic nerve is crushed.

Endothelin-1 (ET-1) causes death of retinal neurons through activation of nitric oxide synthase (NOS) and production of superoxide anion.

Endothelin-1 (ET-1) is the most potent and long-acting vasoconstricting peptide presently known. In addition to its vascular effects, endothelin signaling pathway exists in the central nervous system (CNS), which is deeply related to neuronal degeneration. In the present study, we evaluated the effect of ET-1 on death of retinal neurons consisting mainly of amacrine cells, and its interaction with nitric oxide synthase (NOS) and superoxide production. Cultured retinal neurons from fetal rats were exposed to various doses of ET-1 (0.1, 1.0, 10 and 100nM). Neuronal toxicity of ET-1 was assessed by trypan blue exclusion, Hoechst 33,258 staining and TUNEL assay at different times. Intracellular levels of nitric oxide (NO), superoxide and peroxynitrite were determined semiquantitatively by DAF2-DA, hydroethidine and dihydrorhodamine-123, respectively. The effects of adding SOD (100U/ml) and L-NAME with ET-1 on these changes were evaluated. In addition, the receptor mechanisms involved in these reactions were determined by BQ-123 and BQ-788, receptor antagonists for ET A and ET B receptors, respectively. Exposure of cultured retinal neurons to ET-1 reduced the percentage of living cells in a dose- and time-dependent way, and the percentage of living cells was significantly increased by addition of SOD and L-NAME. Fluorometric analyses revealed that ET-1 increased the intracellular NO level in a dose- and time-dependent manner. The intracellular superoxide and peroxynitrite levels were also significantly increased 24h after incubation with 100nM of ET-1, and this elevation was suppressed by SOD and L-NAME. These ET-1-induced alterations were significantly suppressed when both BQ-123 and BQ-788 were added simultaneously with ET-1 to the medium. These results indicate that the neuronal death caused by ET-1 is most likely mediated by the activation of NOS in association with the formation of superoxides and peroxynitrite.

Angiotensin II receptor blocker inhibits abnormal accumulation of advanced glycation end products and retinal damage in a rat model of type 2 diabetes.

The effects of an angiotensin II receptor blocker (ARB) on the accumulation of one of advanced glycation end products (AGEs), pentosidine, expression of vascular endothelial growth factor (VEGF) and retinal function were investigated in Spontaneously Diabetic Torii (SDT) rats. Candesartan, an ARB, was administered to SDT rats from 10 to 44 weeks of age and the results compared with untreated SDT rats and SD rats. Electroretinograms (ERGs) were recorded to evaluate retinal function. At 44 weeks of age, pentosidine was quantified in the vitreous, lens and plasma using high-performance liquid chromatography (HPLC). Real-time reverse transcription-PCR (RT-PCR) analysis was also performed in order to measure VEGF mRNA expression in the retina. Histological changes were examined and immunohistochemistry for pentosidine performed on the retina and retinal microvasculature. In untreated SDT rats, the amplitudes of a- and b-waves, oscillatory potentials were reduced significantly at 44 weeks of age compared with the 10-week levels, whereas they remained unchanged in SDT rats treated with candesartan. The concentration of pentosidine in the vitreous and lens did not change in treated SDT rats but increased in untreated SDT rats. Retinal VEGF mRNA expression was inhibited in treated SDT rats. Histologically, proliferative tissue was detected around the optic disc, with pentosidine being detected only in untreated SDT rats. Our findings indicate the ARB may inhibit the development of diabetic retinopathy by reducing the accumulation of pentosidine, one of AGEs and expression of VEGF in the retina.

High infusion pressure in conjunction with vitreous surgery alters the morphology and function of the retina of rabbits.

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 = 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.

Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line.

PURPOSE: ARPE-19 is a spontaneously transformed cell line of human RPE that is widely studied. This report examines its suitability for studying the tight junctions of the RPE. METHODS: ARPE-19 was maintained in standard medium or one of three reduced-serum medium formulations. The expression and distribution of cytoskeletal and junctional proteins were examined by immunocytochemistry, immunoblot analysis, and the reverse transcription-polymerase chain reaction. Barrier function was measured as the transepithelial electrical resistance (TER) and the transmonolayer diffusion of horseradish peroxidase (HRP). RESULTS: Unlike the original reports using passage-15 to -20 cells, commonly available strains of ARPE-19 exhibited a heterogeneous mixture of elongate and polygonal cells. Actin was distributed in stress fibers rather than circumferential bands. The TER was low, and the permeability of HRP was high. The expression of claudins and cytokeratins was heterogeneous. Partial differentiation could be induced in subsets of cells by manipulating the growth medium. A common effect was an increase in the expression of JAM-A, AF-6, and PAR-3 that correlated with a redistribution of actin filaments. This effect was accompanied by a 10x decrease in the permeability of HRP, but a minimal effect on TER. CONCLUSIONS: 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.

Expression of JAM-A, AF-6, PAR-3 and PAR-6 during the assembly and remodeling of RPE tight junctions.

The tight junctions of the endothelial and epithelial regions of the blood-brain barrier are regulated by interactions with the neighboring tissue. We examined how the neural retina regulates the assembly of tight junctions in the retinal pigment epithelium (RPE). The proteins JAM-A, AF-6, PAR-3 and PAR-6 have been implicated in the assembly of other epithelial tight junctions. Using chick embryos and primary cell culture, we examined gene expression of these proteins during embryonic development, and whether retinal secretions regulate their expression. Three highly conserved RNA splice sites of AF-6 were identified in chick ocular tissues, but only two were expressed in RPE. JAM-A and AF-6 were expressed at relatively high levels early in development when adherens junctions form, but before tight junctions form. Expression of JAM-A and the AF-6 isoforms actually decreased when tight junctions were forming and expanding. The expression of PAR-3 and PAR-6 was constant. Despite the expression of these proteins in vitro (along with claudins, occludin, ZO-1 and ZO-2), the tight junctional networks that form were discontinuous (Rahner, C., Fukuhara, M., Peng, S., Kojima, S., Rizzolo, L.J., 2004. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J. Cell Sci. 117, 3307-3318). The expression of these assembly proteins was unaffected by a retinal conditioned medium that induced the completion of tight junction formation. These data indicate that the early expression of the assembly proteins corresponds to the initial establishment of the adherens and tight junctions, but secretory products of the neural retina must induce the expression of additional proteins to complete the maturation process.

Endothelin-1 enhances glutamate-induced retinal cell death, possibly through ETA receptors.

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(-10)-10(-7)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 < 0.05, Dunnett), whereas brief exposure of neurons to up to 1 microM 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.

The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development.

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.