Single crystal Er3+ : YAG fibers with tailored refractive index profiles.

Erbium-doped yttrium aluminum garnet (Er3+:YAG) rods were inserted inside undoped tubes and grown into single-crystal fibers of a diameter of 300 µm using the laser-heated pedestal growth technique. Growth at various rates resulted in radially graded distributions of Er3+ dopant ions, as observed using laser-induced fluorescence imaging. Profiles of the refractive index were measured using cross-sectional reflectometry in a microscope. Dopant distributions and the corresponding index profiles were compared with thermal diffusion theory to determine the inter-diffusion coefficient of Y3+ and Er3+ ions at 2000°C, yielding an estimated value of D=(9.10±0.8)×10-11 m2/s. This work constitutes a step toward controlled growth of fibers with high thermal conductivities, low Brillouin gain, and waveguiding properties required for high-power optical amplifier and laser applications.

HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists.

Coat protein gp120 from the human immunodeficiency virus type-1 (HIV-1) increased intracellular free calcium and injured rodent retinal ganglion cells and hippocampal neurons in culture. Highly purified recombinant gp120 envelope protein produced these effects in a dose-dependent fashion at picomolar concentrations. Immunoprecipitation with antibody to gp120, but not with control immunoglobulin-containing serum, depleted solutions of the viral envelope protein and also prevented both the rise in intracellular calcium and neuronal toxicity. The gp120-induced increase in intracellular calcium was abrogated by transiently lowering extracellular calcium or by adding the dihydropyridine calcium channel antagonist nimodipine (100 nM). Calcium channel antagonists also prevented gp120-induced neuronal injury. In addition, intracellular stores appeared to contribute substantially to the increase in calcium elicited by gp120. Since increases in intracellular calcium have been associated with neurotoxicity, it is possible that an injurious effect of gp120 on neurons might be related to this mechanism and that treatment with calcium channel antagonists may prove useful in mitigating HIV-1-related neuronal injury.

Productivity, water-use efficiency and tolerance to moderate water deficit correlate in 33 poplar genotypes from a Populus deltoides x Populus trichocarpa F1 progeny.

Genotypic variability for productivity, water-use efficiency and leaf traits in 33 genotypes selected from an F1 progeny of Populus deltoides Bartr. ex Marsh x Populus trichocarpa L. was explored under optimal and moderate water-deficit conditions. Saplings of the 33 genotypes were grown in a two-plot open field at INRA Orléans (France) and coppiced every year. A moderate water deficit was induced during two successive years on one plot by withholding irrigation, while the second one remained irrigated (control). Stem biomass and leaf structure (e.g., specific leaf area and leaf area) were measured in 2004 and 2005 and functional leaf traits (e.g., carbon isotope discrimination, Delta) were measured only in 2004. Tolerance to water deficit was estimated at genotype level as the ability to limit losses in biomass production in water deficit versus control trees. Stem biomass, leaf structure and Delta displayed a significant genotypic variability whatever the irrigation regime. For all traits, genotype ranks remained stable across years for similar irrigation conditions. Carbon isotope discrimination scaled negatively with productivity and leaf nitrogen content in controls. The most productive genotypes were the least tolerant to moderate water deficit. No relationship was evidenced between Delta and the level of tolerance to water deficit. The relationships between traits evidenced in this collection of P. deltoides x P. trichocarpa F1 genotypes contrast with the ones that were previously detected in a collection of P. deltoides x Populus nigra L. cultivars tested in the same field trial.

Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity.

Exposure of rat retinal cultures to HIV-1 coat protein gp120 for several minutes increases [Ca2+]i in approximately half of the ganglion cells; this effect is associated with delayed-onset neuronal injury, similar to that previously reported in NMDA receptor-mediated neurotoxicity. Here we show that NMDA antagonists can prevent both the rise in [Ca2+]i and subsequent neuronal damage engendered by 20 pM gp120. However, whole-cell patch-clamp recordings demonstrate that gp120 does not directly evoke an NMDA-like response or enhance glutamate/NMDA-activated currents. Moreover, complete protection from gp120-induced [Ca2+]i increases and neurotoxicity is afforded by incubation with glutamate-pyruvate transaminase, which breaks down endogenous glutamate as verified by HPLC. Since, under standard conditions in these cultures, neither glutamate nor a low picomolar concentration of gp120 is deleterious on its own, our results suggest that their neurotoxicity is synergistic.

Responses of Antioxidative Systems to Drought Stress in Pendunculate Oak and Maritime Pine as Modulated by Elevated CO2.

The aim of the present study was to investigate the effects of an enhanced CO2 concentration alone or in combination with drought stress on antioxidative systems of a deciduous (oak; Quercus robur) and an evergreen (pine; Pinus pinaster) tree species. The seedlings were grown for one season in a greenhouse in tunnels supplied with 350 or 700 [mu]L L-1 CO2. The experiment was repeated in a second year. Antioxidants, protective enzymes, soluble protein, and pigments showed considerable fluctuations in different years. Elevated CO2 caused significant reductions in the activities of superoxide dismutases in both oak and pine. The activities of ascorbate peroxidase and catalase were also reduced in most cases. The activities of dehydroascorbate reductase, monodehydroascorbate radical reductase, glutathione reductase, and guaiacol peroxidase were affected little or not at all by elevated CO2. When the trees were subjected to drought stress by withholding water, the activities of antioxidative enzymes decreased in leaves of pine and oak grown at ambient CO2 and increased in plants grown at elevated CO2 concentrations. The present results suggest that growth in elevated CO2 might reduce oxidative stress to which leaf tissues are normally exposed and enhance metabolic flexibility to encounter increased stress by increases in antioxidative capacity.

bcl-2 gene therapy exacerbates excitotoxicity.

The protooncogene bcl-2 can block neuronal death from both naturally occurring apoptosis and exogenous insults. bcl-2 is therefore a promising candidate for the prevention of excitotoxic neuronal death. Using an adeno-associated viral vector, we delivered the bcl-2 gene to the ganglion cell layer of the rat eye. We hypothesized that infection with bcl-2 would protect ganglion cells against excitotoxic cell death. However, retinal infection with bcl-2 increased ganglion cell susceptibility to both axonal injury and intravitreal NMDA. Our study--intended to explore the possibility of bcl-2 transduction as an in vivo therapeutic approach--revealed a deleterious effect of bcl-2 transduction.

Concurrent downregulation of a glutamate transporter and receptor in glaucoma.

PURPOSE: Elevated levels of extracellular glutamate have been implicated in the pathophysiology of neuronal loss in both central nervous system and ophthalmic disorders, including glaucoma. This increase in glutamate may result from a failure of glutamate transporters, which are molecules that ordinarily regulate extracellular glutamate. Elevated glutamate levels can also lead to a perturbation in glutamate receptors. The hypothesis for the current study was that glutamate transporters and/or receptors are altered in human glaucoma. METHODS: Immunohistochemical analyses of human eyes with glaucoma and control eyes were performed to evaluate glutamate receptors and transporters. These molecules were also assayed in rat eyes injected with glial-derived neurotrophic factor (GDNF). RESULTS: Glaucomatous eyes had decreased levels of both the glutamate transporter, excitatory amino acid transporter (EAAT)-1, and the glutamate receptor subunit N-methyl-D-aspartate (NMDA)-R1. Eyes treated with GDNF had elevated levels of both EAAT1 and NMDAR1. CONCLUSIONS: The loss of EAAT1 in glaucoma may account for the elevated level of glutamate found in glaucomatous vitreous and lead to a compensatory downregulation of NMDAR1. Inasmuch as GDNF can increase levels of both EAAT1 and NMDAR1, it may be a useful therapeutic approach to restore homeostatic levels of these in glaucoma.

Retinal Thy-1 expression during development.

PURPOSE: To evaluate the developmental expression of Thy-1 in the retina. Thy-1, the most abundant mammalian neuronal surface glycoprotein, is likely to play a significant role in retinal development. In the mammalian retina, it is found predominantly, if not exclusively, on retinal ganglion cells. METHODS: Rat retinae of various ages were stained immunohistochemically for Thy-1 with 2G12, a monoclonal Thy-1 antibody. Sections were analyzed digitally to quantify bound antibody. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the expression of Thy-1 protein was compared with the levels of mRNA detected. RESULTS: Thy-1-dependent fluorescence was detected in rat retinae from birth, albeit at low levels. Thy-1 labeling was localized predominantly to the ganglion cell layer. Minimal, fine patterns of linear and reticular fluorescence were noted in the inner nuclear layer. Thy-1 levels reached a maximal level at approximately postnatal day 14. RT-PCR measurements showed a similar time course for the increase in Thy-1 expression. CONCLUSIONS: The Thy-1 antigen is present in the inner retina at birth. Its level increases steadily after birth and peaks during the second week of life. Thy-1 expression is approximately coterminous with synaptogenesis of the inner plexiform layer and may play a role in synaptogenesis of the inner retina or in other developmental milestones in the formation of the visual system.

Ganglion cell loss after optic nerve crush mediated through AMPA-kainate and NMDA receptors.

PURPOSE: Glutamate antagonists can block ganglion cell death due to optic nerve crush. Although most investigators have focused on blockade of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, we have chosen to evaluate the efficacy of blockade of the AMPA-kainate (KA) receptor in this experimental paradigm. METHODS: The optic nerves of rats were crushed, and ganglion cell survival was assessed. Groups of animals were treated with an NMDA antagonist, an AMPA-KA antagonist, or both. RESULTS: The AMPA-KA antagonist DNQX was more effective, although not additive in preserving retinal ganglion cells after optic nerve crush than the NMDA antagonist MK801. CONCLUSIONS: Activation of the AMPA-KA subtype of glutamate receptor may play a role in glutamate-mediated cell death after optic nerve crush.

Systemic L-kynurenine administration partially protects against NMDA, but not kainate-induced degeneration of retinal ganglion cells, and reduces visual discrimination deficits in adults rats.

PURPOSE: Kynurenic acid (KYNA), an endogenous tryptophan metabolite, is an N-methyl-D-aspartate (NMDA) antagonist active at the glycine-binding site of the NMDA-receptor complex. The authors investigated whether systemic administration of a biochemical precursor of KYNA, L-kynurenine (L-Kyn), could block NMDA- or kainic acid (KA)-induced cell death in adult rat retinal ganglion cells (RGCs) and protect NMDA-treated animals from lesion-induced visual deficits. METHODS: Rats were injected with 20-nmol NMDA or 5-nmol KA intraocularly. To quantify the number of surviving RGCs, the retrograde tracer horseradish-peroxidase was injected into the superior colliculus contralateral to the lesioned eye. Surviving RGCs were counted on wholemounted retinae in a centroperipheral gradient, as well as in the four quadrants, using a computer-assisted image analysis system. RESULTS: The NMDA-injections resulted in an approximately 82% RGC loss in the adult rat retina compared with control retinae and a cell loss of approximately 50% in KA-treated retinae. Pretreatment with L-Kyn significantly reduced NMDA-induced RGC degeneration to values of approximately 60%, but KA toxicity was not significantly affected by L-Kyn pretreatment. Intraocular injections of NMDA resulted in an impairment of visual discrimination behavior, which partially recovered within a period of approximately 3 weeks. However, when treated systemically with L-Kyn, brightness discrimination was significantly improved as compared with NMDA-treated rats. CONCLUSIONS: These findings show that systemic administration of L-Kyn in adult rats can block NMDA-induced retinal ganglion cell death in vivo and preserves brightness discrimination performance.

Ethambutol is toxic to retinal ganglion cells via an excitotoxic pathway.

PURPOSE: Ethambutol is an essential medication in the management of tuberculosis. However, it can cause an optic neuropathy of uncertain etiology. Ethambutol toxicity was therefore studied in rodent retinal cells, and agents that might block its toxicity were considered. METHODS: The toxicity of ethambutol and related agents was evaluated in rodent retinal dissociated cell preparations and whole eyes. Calcium fluxes and mitochondrial function were evaluated by fluorescent and staining techniques. For in vivo assays, adult rats were administered oral ethambutol over a 3-month period. Cell survival was assessed by stereology. RESULTS: Ethambutol is specifically toxic to retinal ganglion cells in vitro and in vivo. Endogenous glutamate is necessary for the full expression of ethambutol toxicity, and glutamate antagonists prevent ethambutol-mediated cell loss. Ethambutol causes a decrease in cytosolic calcium, an increase in mitochondrial calcium, and an increase in the mitochondrial membrane potential. CONCLUSIONS: The visual loss associated with ethambutol may be mediated through an excitotoxic pathway, inasmuch as ganglion cells are rendered sensitive to normally tolerated levels of extracellular glutamate. Ethambutol perturbs mitochondrial function. Its toxicity may depend on decreased ATPase activity and mitochondrial energy homeostasis. Glutamate antagonists may be useful in limiting the side effects seen with ethambutol.

The contribution of various NOS gene products to HIV-1 coat protein (gp120)-mediated retinal ganglion cell injury.

PURPOSE: There is growing evidence that the neuronal pathology seen with HIV-1 is mediated, at least in part, through an excitotoxic/free radical pathway. Nitric oxide (NO) plays a critical role in the nervous system, in both normal and pathologic states, and appears to be involved in a variety of excitotoxic pathways. Whether isoforms of nitric oxide synthase (NOS) are involved in gp120-mediated neuronal loss in the retina was therefore explored. METHODS: To determine which (if any) of the various isoforms of NOS are critical in gp120-mediated damage in the retina, neuronal NOS-deficient [nNOS(-/-)], endothelial NOS-deficient [eNOS(-/ -)], and immunologic NOS-deficient [iNOS(-/-)] mice were subjected to intravitreal injections of gp120. RESULTS: Retinal ganglion cells in the nNOS(-/-) mouse were relatively resistant to gp120, manifesting attenuation of gp120-induced injury compared with wild-type mice. The iNOS(-/-) and eNOS(-/-) mice were as susceptible to gp120 toxicity as control animals. NOS inhibitors were protective against this toxicity. CONCLUSIONS: The presence of nNOS is a prerequisite for the full expression of gp120-mediated loss in the retina; eNOS and iNOS do not appear to play a significant role.

Chronic low-dose glutamate is toxic to retinal ganglion cells. Toxicity blocked by memantine.

PURPOSE: It is well known that acute exposure to high concentrations of glutamate is toxic to central mammalian neurons. However, the effect of a chronic, minor elevation over endogenous glutamate levels has not been explored. The authors have suggested that such chronic exposure may play a role in glaucomatous neuronal loss. In the current study, they sought to explore whether a chronic, low-dose elevation in vitreal glutamate was toxic to retinal ganglion cells and whether this toxicity could be prevented with memantine, a glutamate antagonist. METHODS: Rats were injected serially and intravitreally with glutamate to induce chronic elevations in glutamate concentration. A second group of rats was treated with intraperitoneal memantine and glutamate. Control groups received vehicle injection with or without concurrent memantine therapy. After 3 months, the animals were killed, and ganglion cell survival was evaluated. RESULTS: Intravitreal injections raised the intravitreal glutamate levels from an endogenous range of 5 to 12 microM glutamate to 26 to 34 microM. This chronic glutamate elevation killed 42% of the retinal ganglion cells after 3 months. Memantine treatment alone had no effect on ganglion cell survival. However, when memantine was given concurrently with low-dose glutamate, memantine was partially protective against glutamate toxicity. CONCLUSIONS: These data suggest that minor elevations in glutamate concentration can be toxic to ganglion cells if this elevation is maintained for 3 months. Furthermore, memantine is efficacious at protecting ganglion cells from chronic low-dose glutamate toxicity.

Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death.

PURPOSE: Elevated levels of extracellular glutamate have been implicated in the pathophysiology of neuronal loss in both central nervous system and ophthalmic disorders, including glaucoma. This increase in glutamate may result from a failure of glutamate transporters (molecules that ordinarily regulate extracellular glutamate; E:xcitatory A:mino A:cid T:ransporter; EAAT). Elevated glutamate levels can also lead to alterations in glutamate receptor expression. It was hypothesized that selective blockade of glutamate transporters would be toxic to retinal ganglion cells. METHODS: Glutamate transporters were blocked either pharmacologically or with subtype-specific antisense oligonucleotides against EAAT1. Glutamate levels, transporter levels and ganglion cell survival were assayed. RESULTS: Pharmacological inhibition of glutamate transporters with either an EAAT2 specific inhibitor or a nonspecific inhibitor of all the subtypes of transporters was toxic to ganglion cells. Treatment with oligonucleotides against the glutamate transporter EAAT1 decreased the levels of expression of the transporter, increased vitreal glutamate, and was toxic to ganglion cells. CONCLUSIONS: These results demonstrate that normal function of EAAT1 and EAAT2 is necessary for retinal ganglion cell survival and plays an important role in retinal excitotoxicity. Manipulation of retinal glutamate transporter expression may become a useful tool in understanding retinal neuronal loss.

The role of neuronal and endothelial nitric oxide synthase in retinal excitotoxicity.

PURPOSE: Nitric oxide synthase (NOS) plays an essential role in neuronal function and is critical in the brain for normal and pathologic responses to glutamate. The role of NOS in the retina is less well understood. The retina provides an experimental system in which the intrinsic circuitry is well defined; retinal excitotoxic damage has been well characterized. METHODS: To determine whether neuronal NOS (nNOS) and endothelial NOS (eNOS) are critical in excitotoxic damage in the retina, nNOS- and eNOS-deficient mice were subjected to intravitreal injections of N-methyl-D-aspartate (NMDA) or to arterial occlusions. RESULTS: Retinal ganglion cells in the nNOS-deficient mouse were relatively resistant to NMDA and to arterial occlusion. In contrast, the damage in the eNOS-deficient mouse retina was not distinguishable from that in control animals. Preinjection with an NOS inhibitor was partially protective. CONCLUSIONS: The presence of nNOS is a prerequisite for the full expression of excitotoxicity in the retina; eNOS does not appear to play a significant role.

Pilocarpine toxicity in retinal ganglion cells.

PURPOSE: Muscarinic agents reduce intraocular pressure by enhancing aqueous outflow, probably by stimulating ciliary muscle contraction. However, pilocarpine is a well characterized neurotoxin and is widely used to generate animal seizure models. It was therefore investigated whether pilocarpine was also toxic to retinal ganglion cells. METHODS: Dissociated whole retinal preparations were prepared from postnatal day 16 to 19 rats. Retinal ganglion cells had been previously back-labeled with a fluorescent tracer. Retinal cells were incubated with pilocarpine, lithium, and inositol derivatives, and viability of the retrogradely labeled retinal ganglion cells was assayed after 24 hours. RESULTS: Pilocarpine was toxic to retinal ganglion cells in a dose-dependent fashion. This toxicity was potentiated by lithium and blocked by epi- and myo-inositol. CONCLUSIONS: Pilocarpine is toxic to retinal ganglion cells in a mixed culture assay. This toxicity appears to depend on the inositol pathway and is similar to its mode of action in other neurons. However, 0.4 mM pilocarpine (the lowest concentration that did not affect ganglion cell survival) is roughly 1000-fold higher than the vitreal concentration and 20-fold higher than the scleral concentration that can be obtained with topical administration of 2% pilocarpine in the rabbit eye.

Effect of mitomycin C and fluorouracil-supplemented trabeculectomies on the anterior segment.

OBJECTIVES: To determine whether there is increased risk to the corneal endothelium when mitomycin C is used in trabeculectomy surgery instead of fluorouracil, and whether these agents play a role in accelerating cataract formation. DESIGN, SETTINGS, AND PARTICIPANTS: We analyzed the corneal endothelium and the lens preoperatively and postoperatively in 30 eyes of 21 patients who underwent either a fluorouracil- or mitomycin C-supplemented trabeculectomy. RESULTS: No significant differences were found between these two groups in the rate of cataract progression, magnitude of endothelial cell loss, or appearance of endothelial cell morphologic characteristics. Endothelial cell loss accounted for approximately 7% to 8% of the preoperative counts in both groups. In addition, four (27%) of 15 eyes in each group showed evidence of cataract progression as graded by the Lens Opacities Classification System II. CONCLUSION: Fluorouracil- and mitomycin C-supplemented trabeculectomies cause similar changes in the lens and corneal endothelium.

Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma.

OBJECTIVE: To explore the possibility that the excitatory amino acid glutamate might be associated with the disease process of glaucoma, which is characterized by the death of retinal ganglion cell neurons and subsequent visual dysfunction. METHODS: Amino acid analyses were performed on vitreous specimens that were obtained from patients who were undergoing cataract extraction. Samples were collected prospectively from those patients who sustained inadvertent rupture of the posterior capsule between 1988 and 1993. An additional set of specimens, obtained from both eyes of monkeys, was analyzed; in these monkeys, glaucoma had been experimentally induced in one eye only. RESULTS: A twofold elevation in the level of glutamate was detected in the vitreous body of the group of patients with glaucoma when compared with that in a control population of patients with cataracts only. An even greater elevation of the glutamate level was found in the vitreous body of glaucomatous eyes of monkeys when compared with that in control eyes. No statistical differences were detected among other amino acid levels from the vitreous body of glaucomatous and nonglaucomatous eyes in humans or monkeys. CONCLUSIONS: The excitatory amino acid glutamate is found in the vitreous body of glaucomatous eyes at concentrations that are potentially toxic to retinal ganglion cells. The increased level of this known neurotoxin is consistent with an "excitotoxic" mechanism for the retinal ganglion cell and optic nerve damage in glaucoma. Therapies to protect neurons against glutamate toxic effects may prove to be useful in the management of this blinding disease.

New horizons in neuroprotection.

Glaucoma is a leading cause of blindness worldwide and the second leading cause of irreversible blindness in the USA. The most common form of glaucoma, primary open angle glaucoma, is characterized by a chronically elevated intraocular pressure in the absence of any demonstrable structural abnormalities in the eye. The pathologic hallmark of glaucomatous optic neuropathy is the selective death of retinal ganglion cells associated with structural changes in the optic nerve head. Recent discoveries suggest a role for nitric oxide, glutamate, apoptosis, and others, in the pathophysiology of this neuropathy. These newer discoveries are addressed in this article.

An experimental basis for implicating excitotoxicity in glaucomatous optic neuropathy.

Most therapy for glaucoma is directed at the management of the intraocular pressure (IOP). Conventional wisdom holds that excessive pressure within the eye leads to the ganglion cell loss/optic nerve damage seen in this disease. Both glutamate and elevated IOP can selectively damage the retinal ganglion cells in the mammalian eye. We have identified an elevated level of glutamate in the vitreous humor of glaucoma patients (27 microM as compared to 11 microM in the control population). This concentration of glutamate suffices--on its own--to kill retinal ganglion cells. It is plausible that the IOP may represent an initial insult that precipitates the production of excessive glutamate. Therefore, even if glutamate elevation is an epiphenomenon associated with the course of the disease, it may contribute to ganglion cell loss in humans. Lowering the IOP may slow down glutamate production, but if nothing is done to block the toxic effects of glutamate as well, visual loss may result despite excellent IOP control. If interventions can be found to retard the production or toxic effects of glutamate, it may be possible to slow glaucomatous visual loss.