Light-induced charge generation in polymeric nanoparticles restores vision in advanced-stage retinitis pigmentosa rats.

Retinal dystrophies such as Retinitis pigmentosa are among the most prevalent causes of inherited legal blindness, for which treatments are in demand. Retinal prostheses have been developed to stimulate the inner retinal network that, initially spared by degeneration, deteriorates in the late stages of the disease. We recently reported that conjugated polymer nanoparticles persistently rescue visual activities after a single subretinal injection in the Royal College of Surgeons rat model of Retinitis pigmentosa. Here we demonstrate that conjugated polymer nanoparticles can reinstate physiological signals at the cortical level and visually driven activities when microinjected in 10-months-old Royal College of Surgeons rats bearing fully light-insensitive retinas. The extent of visual restoration positively correlates with the nanoparticle density and hybrid contacts with second-order retinal neurons. The results establish the functional role of organic photovoltaic nanoparticles in restoring visual activities in fully degenerate retinas with intense inner retina rewiring, a stage of the disease in which patients are subjected to prosthetic interventions.

Retinal long term neuroprotection by Cerium Oxide nanoparticles after an acute damage induced by high intensity light exposure.

Cerium Oxide nanoparticles are antioxidant agents with autoregenerative radical scavenging activities, effective in preventing degeneration of photoreceptors of an albino rat when intravitreally injected prior to exposure to high intensity light. In this study, we performed a post injury administration of nanoceria and a long term analysis of their neuroprotective properties in order to better simulate the therapeutic treatment as it is carried out on patients with age related macular degeneration, and while photoreceptor degeneration is ongoing. We also injected nanoceria labelled with fluorescein isothiocianate in order to analyze their persistence after a single administration in a damaged retina and to investigate how long they both maintain their neuroprotective properties and where they localize in the retina. We demonstrated that after a single intravitreal injection, nanoceria remained in the retina for a long time and retained their neuroprotective properties. All these data form excellent bases for future clinical applications.

A longitudinal follow-up study of saffron supplementation in early age-related macular degeneration: sustained benefits to central retinal function.

Objectives. In a previous randomized clinical trial (Falsini et al. (2010)), it was shown that short-term Saffron supplementation improves retinal flicker sensitivity in early age-related macular degeneration (AMD). The aim of this study was to evaluate whether the observed functional benefits from Saffron supplementation may extend over a longer follow-up duration. Design. Longitudinal, interventional open-label study. Setting. Outpatient ophthalmology setting. Participants. Twenty-nine early AMD patients (age range: 55-85 years) with a baseline visual acuity >0.3. Intervention. Saffron oral supplementation (20 mg/day) over an average period of treatment of 14 (±2) months. Measurements. Clinical examination and focal-electroretinogram-(fERG-) derived macular (18°) flicker sensitivity estimate (Falsini et al. (2010)) every three months over a followup of 14 (±2) months. Retinal sensitivity, the reciprocal value of the estimated fERG amplitude threshold, was the main outcome measure. Results. After three months of supplementation, mean fERG sensitivity improved by 0.3 log units compared to baseline values (P < 0.01), and mean visual acuity improved by two Snellen lines compared to baseline values (0.75 to 0.9, P < 0.01). These changes remained stable over the follow-up period. Conclusion. These results indicate that in early AMD Saffron supplementation induces macular function improvements from baseline that are extended over a long-term followup.

The impact of organic inhibitors of the hyperpolarization activated current (Ih) on the electroretinogram (ERG) of rodents.

We have compared the effect of two distinct Ih inhibitors on the temporal properties of the ERG response that, as previously shown, correlates well with the HCN activation in rods. The present results confirm the notion that cilobradine is more effective than zatebradine in inducing bradycardia. Importantly, the doses of cilobradine that reduce the heart rate to values comparable to, or lower than, those obtained with higher doses of zatebradine have little effect on the frequency response of the ERG. While more potent than zatebradine in its bradycardic action, cilobradine appears comparatively less effective on the visual response. A possible explanation is that the affinity of cilobradine for the HCN channels in the heart is higher than that for the HCN channels of retinal neurons.

Electroretinogram changes associated with retinal upregulation of trophic factors: observations following optic nerve section.

The purpose of the present work was to assess whether upregulation of trophic factors and protection from damage induced in the retina by optic nerve section are associated with changes in the flash electroretinogram (ERG). We have examined the ERG in adult pigmented rat at different survival times over a period of 3 months following section of the optic nerve. The a-wave was analyzed using the Lamb-Pugh model and the parameters of best fit were estimated in control animals and at successive survival times. The amplitudes of the a- and b-waves were reduced over the first 7 days after nerve section. The a-wave recovered its relative amplitude by 21 days, but the b-wave remained depressed 5 weeks following nerve section. Analysis of the a-wave indicated a 20-30% reduction in the dark current of sectioned eyes at 7 days survival. A significant reduction of the amplification constant was observed in both nerve-sectioned and nerve-intact eyes, relative to normal and sham-operated controls. This reduction persisted to the longest survival time examined. The reduction of the a-wave at 7 days after nerve section coincides with a period of upregulation of ciliary nerve trophic factor. The amplification factor is influenced over a longer time course, which corresponds with a period of up-regulation of basic fibroblast growth factor. These changes in growth factor expression and ERG parameters are in turn associated with protection of photoreceptors against light damage. Present results suggest that the sensitivity of the retina to light may be regulated by mechanisms which protect photoreceptors against stress.

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project.

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g. increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.

The outcome of Leishmania major experimental infection in BALB/c mice can be modulated by exogenously delivered iron.

We previously established that Leishmania promastigotes express a transferrin receptor and that iron chelators inhibit promastigote growth in vitro. Thus, we were interested in modulating the vertebrate host iron pool and to monitor whether such changes will affect the outcome of L. major infection in BALB / c mice, inoculated in the footpad with 106 stationary phase promastigotes. Treatment of mice with desferrioxamine resulted in a slight delay of the development of cutaneous lesions. In contrast and unexpectedly, systemic iron delivery, at early time points of parasite delivery, significantly limited footpad pathology. Accordingly, parasite loads at the site of parasite delivery, the draining lymph node, liver and spleen were significantly reduced in iron-loaded mice. Importantly, the "protective" effect of iron delivery correlated with the presence, at the site of inoculation, of lower levels of IL-4 and IL-10 transcripts while both IFN-gamma and inducible nitric oxide synthase transcripts were at higher levels. The presence of more type 1 cytokine transcripts was further supported by the increased levels of IgG2a in their sera. These data strongly suggest that susceptibility to L. major as assessed in the footpad model is modifiable by interventions that alter the iron status of the host at early time points of parasite delivery.

Mechanisms of photoreceptor death and survival in mammalian retina.

The mammalian retina, like the rest of the central nervous system, is highly stable and can maintain its structure and function for the full life of the individual, in humans for many decades. Photoreceptor dystrophies are instances of retinal instability. Many are precipitated by genetic mutations and scores of photoreceptor-lethal mutations have now been identified at the codon level. This review explores the factors which make the photoreceptor more vulnerable to small mutations of its proteins than any other cell of the body, and more vulnerable to environmental factors than any other retinal neurone. These factors include the highly specialised structure and function of the photoreceptors, their high appetite for energy, their self-protective mechanisms and the architecture of their energy supply from the choroidal circulation. Particularly important are the properties of the choroidal circulation, especially its fast flow of near-arterial blood and its inability to autoregulate. Mechanisms which make the retina stable and unstable are then reviewed in three different models of retinal degeneration, retinal detachment, photoreceptor dystrophy and light damage. A two stage model of the genesis of photoreceptor dystrophies is proposed, comprising an initial "depletion" stage caused by genetic or environmental insult and a second "late" stage during which oxygen toxicity damages and eventually destroys any photoreceptors which survive the initial depletion. It is a feature of the model that the second "late" stage of retinal dystrophies is driven by oxygen toxicity. The implications of these ideas for therapy of retinal dystrophies are discussed.

Temporal fidelity in the visual system.

The temporal properties of the visual system have been analyzed by recording the ERG and its isolated components in response to light stimuli whose luminance was varied sinusoidally. The performance of the visual system to periodic light stimuli was tested in human subjects psychophysically. The comparison of the results in control conditions and after administration of drugs that specifically block the hyperpolarization activated current (Ih) suggest that the inner rectifying properties of the inner segment membrane of rods is involved in a process of temporal differentiation of the visual signals whereby high frequency components of the response especially relevant for the visual performance are enhanced. It is proposed that the temporal fidelity of the visual system is the results of an elaboration starting at early level of the signal generated by the phototransductive cascade.

The impact of basic fibroblast growth factor on photoreceptor function and morphology.

PURPOSE: To assess the impact of basic fibroblast growth factor (bFGF) on photoreceptor function and morphology. METHODS: Impact was assessed in two models. In one, the endogenous expression of bFGF in photoreceptors was raised by sectioning one optic nerve of rats 3 to 4 weeks before study. In the other, bFGF was injected into the vitreous chamber in rats and cats. Retinal function was assessed from the electroretinogram (ERG), and retinal morphology was studied using DNA dyes, immunolabeling, and in situ hybridization. RESULTS: In both models of bFGF upregulation, the ERG b-wave was suppressed over a wide stimulus range and in light- and dark-adapted conditions. The a-wave was not suppressed by either procedure and at the brightest intensities was enhanced by both procedures. In nerve-sectioned eyes, outer retina appeared normal histologically, but levels of bFGF protein in the inner and outer nuclear layers were raised, whereas bFGF mRNA levels remained unchanged. In both models, levels of synaptophysin in the outer plexiform layer and of cytochrome oxidase in inner segments were raised in association with increases in bFGF protein levels. CONCLUSIONS: bFGF increased the ability of photoreceptors to respond to light but attenuated the transmission of this response to inner retinal cells, presumably by blocking the photoreceptor-bipolar synapse. If the expression of bFGF protein is upregulated in human photoreceptor dystrophies, it may contribute a reversible component to the loss of vision. The relationship between these actions of bFGF and its ability to protect photoreceptors from stress remains to be established.

Analysis of pharmacologically isolated components of the ERG.

An harmonic analysis was applied to the electroretinogram (ERG) measured in intact cat eyes in control conditions and after pharmacological isolation of the components attributed to photoreceptors (PIII) and bipolar neurons (PII). The frequency response curves obtained in various conditions showed that the bandwidth of the PII component extends over a range of stimulus frequencies higher than the bandwidth of PIII. The enhancement of the PII response to stimuli of high temporal frequency suggests the presence of a frequency dependent gain control located either pre- and/or post-synaptically in the transmission line between the phototransductive cascade and bipolar neurons. A possible role of these processes is to enhance relevant visual information whilst selectively attenuating low frequency signals originating in the transductive cascade.

Effects of blocking the hyperpolarization-activated current (Ih) on the cat electroretinogram.

The temporal properties of the electroretinogram (ERG) recorded from cat eyes were analyzed in the presence of either Cs+ or zatebradine which are known to inhibit the hyperpolarization activated current (Ih) in retinal rods. Both Cs+ and zatebradine reduce the ERG response to high-frequency sinusoidal stimuli of high mean luminance and contrast. Conversely, blockade of Ih has no effect on the frequency response characteristics of the isolated receptor component (PIII). These observations support the idea that Ih plays an important role in the transfer of signals from photoreceptors to second order neurons by suppressing the slow components originated in the phototransductive cascade. The result of this operation is an enhancement of the light response in a range of temporal frequencies relevant to vision.

Electrical activity regulates dendritic reorganization in ganglion cells after neonatal retinal lesion in the cat.

During the first month of postnatal life, the dendritic arborizations of cat retinal ganglion cells continue to develop and undergo a substantial remodeling. Mechanical and pharmacological interferences with the normal development induce, during this period of time, substantial modifications in ganglion cell morphology. Specifically, the degeneration of those neurons whose axons were severed by a neonatal retinal lesion leads to a zone depleted of ganglion cells. Neurons at the border of the depleted area develop an abnormal elongation of the dendritic trees toward the empty space. In the present paper, we report data showing that this dendritic reorganization can be prevented by blocking the electrical activity with repeated tetrodotoxin injections into the eye during the whole critical period. Our analysis was performed on neurons filled with horseradish peroxidase.

Evidence that the influence of ganglion cell axons on astrocyte morphology is mediated by action spike activity during development.

In many mammal retinas, the morphology of astrocytes is strongly influenced by nearby axons of ganglion cells. Astrocyte processes stretch along the axons, fine extensions of the processes contact node-like specialisation of the axon membrane and the morphology of the adult astrocyte is strongly determined by this relationship. The mechanism which attracts astrocyte processes to contact specific regions of the axon membrane is not known however. This study presents evidence that in the neonatal cat blocking the impulse activity of ganglion cells with the Na+-channel blocker tetrodotoxin (TTX) leads to a loss of the axon-related morphology of astrocytes. The morphological change induced in astrocytes by TTX was greater in younger animals and could not be detected in the adult. Conversely, if the TTX block was maintained for 4 postnatal weeks the changes induced in astrocytes persisted at least to 13 weeks. The TTX-induced loss of axon-related morphology in astrocytes suggests that the signal by which axons attract astrocyte processes to contact the axonal membrane in ways which modify astrocyte morphology is released by action spike activity during development.

Blockade of glutamate-mediated activity in the developing retina perturbs the functional segregation of ON and OFF pathways.

The dendrites of ganglion cells initially ramify throughout the inner plexiform layer of the developing retina before becoming stratified into ON or OFF sublaminae. This ontogenetic event is thought to depend on glutamate-mediated afferent activity, because treating the developing retina with the glutamate analog 2-amino-4-phosphonobutyrate (APB), which hyperpolarizes ON cone bipolar cells and rod bipolar cells, thereby preventing their release of glutamate, effectively arrests the dendritic stratification process. To assess the functional consequences of this manipulation, extracellular recordings were made from single cells in the A laminae of the dorsal lateral geniculate nucleus and from the optic tract in mature cats that had received intraocular injections of APB during the first postnatal month. Such recordings revealed that stimulation of the APB-treated eye evoked both ON as well as OFF discharges in 37% of the cells tested. (As expected, when the normal eye was activated, virtually all cells yielded only ON or OFF responses.) The proportion of ON-OFF cells found here corresponds closely to the incidence of multistratified dendrites observed previously in anatomical studies of APB-treated cat retinas. This suggests that the ganglion cells with multistratified dendrites receive functional inputs from ON as well as OFF cone bipolar cells. This interpretation is further supported by the finding that the proportion of ON-OFF cells was very similar in the geniculate layer innervated by the treated eye and in the optic tract. The cells activated by the APB-treated eye were also found not to show response suppression when flashing stimuli of increasing size were used. This suggests that exposing the developing retina to APB perturbs the neural circuitry mediating the antagonistic center-surround organization found in normal receptive fields. The functional changes evident after treating the developing retina with APB suggest that it should now be feasible to assess how the segregation of ON and OFF retinal pathways relates to organizational features at higher levels of the visual system, such as orientation selectivity in cortical cells.

Normal EOG values in intraretinal metastasis from cutaneous melanoma: a case report.

PURPOSE: The electro-oculogram (EOG) is a powerful test to diagnose primary and metastatic choroidal tumors. While in benign tumors light-peak to dark-trough ratio values are in the range of normal subjects, these values appear highly altered in eyes affected by malignant choroidal tumors. Here we report a clinical case of a patient with intraretinal metastasis from cutaneous melanoma; notwithstanding the malignancy of the tumor, the EOG doesn't present alterations. METHODS AND RESULTS: Standard electro-oculographic recordings were performed before and after local excision of the tumor: recordings from the normal eye were taken as control. The EOG values were always normal in both eyes. Histological sections showed no evident change in the cell population of the retinal pigment epithelium (RPE) and Bruch's membrane. CONCLUSION: Our results suggest that the presence of an intact RPE is a crucial requirement to obtain a normal EOG.

Functional development of intrinsic properties in ganglion cells of the mammalian retina.

Senosory neurons manifest pronounced changes in excitability during maturation, but the factors contributing to this ubiquitous developmental phenomenon are not well understood. To assess the contribution of intrinsic membrane properties to such changes in excitability, in the present study whole cell patch-clamp recordings were made from developing ganglion cells in the intact retina of postnatal rats. During a relatively brief developmental period (postnatal days P7-P27) ganglion cells exhibited pronounced changes in the discharge patterns generated by depolarizing current injections. The youngest cells (P7-P17) typically responded to maintained depolarizations with only a single spike or a rapidly adapting discharge pattern. In contrast, the predominant response mode of more mature cells (P21-P27) was a series of repetitive discharges that lasted for the duration of the depolarization period, and by P25 all cells responded in this manner. These functional changes characterized all three morphologically defined cell classes identified by intracellular labeling with Lucifer yellow. To determine if expression of the potassium current (Ia) and the kinetics of the Na-channel related to the increased excitability of developing ganglion cells described above, current- and voltage-clamp recordings were made from individual neurons. The different firing patterns manifested by developing retinal ganglion cells did not reflect the presence or absence of the Ia conductance, although cells expressing Ia tended to generate spikes of shorter duration. With maturation the speed of recovery from inactivation of the Na current increased markedly and this related to the increased excitability of developing ganglion cells. Neurons yielding only a single spike to maintained depolarization were characterized by the slowest speed of recovery; cells with rapidly adapting discharges showed a faster recovery and those capable of repetitive firing recovered fastest from Na-channel inactivation. It is suggested that these changes in intrinsic membrane properties may relate to the different functional roles subserved by ganglion cells during development.

Fate of DNA from retinal cells dying during development: uptake by microglia and macroglia (Müller cells).

The tunel technique of labelling fragmenting dna was used to examine cell death in the developing retina of the rabbit, rat and cat. TUNEL-labelled structures included the still-intact nuclei of retinal cells and smaller, strongly labelled bodies interpreted as fragments of disintegrating nuclei (apoptotic or pyknotic bodies). With confocal microscopy, the cytoplasm around labelled nuclei was observed to be labelled, suggesting that DNA fragments spread into the cytoplasm of the dying cell. Also observed were cells whose nuclei were TUNEL-but whose cytoplasm was TUNEL+, so that their morphology could be discerned. Evidence is presented that these are phagocytes, their cytoplasmic labelling resulting from the ingestion of the fragmenting DNA of a dying neighbour. Results suggest that in developing retina fragmenting DNA is phagocytosed principally by microglia and Müller cells, with a few neurones and no astrocytes active as phagocytes. In the postnatal material studied, microglia are the predominant phagocytes for cells dying in the ganglion cell and inner nuclear layers. Müller cells appear able to phagocytose cells dying in any retinal layer and, since microglia do not normally enter the outer nuclear layer, may be important for the phagocytosis of dying photoreceptors.

Effect of blocking the Na+/K+ ATPase on Ca2+ extrusion and light adaptation in mammalian retinal rods.

Membrane current and light response were recorded from rods of monkey and guinea pig by means of suction electrodes. The correlation between adaptation and the Na+/K+ pump was investigated by measuring light-dependent changes in sensitivity with and without inhibition of Na+/K+ ATPase by strophanthidin. Strophanthidin was found to reduce the dark current, to slow the time course of the photoresponse, and to increase light sensitivity. At concentrations between 20 and 500 nM, the pump inhibitor suppressed in a reversible way the current re-activation occurring during prolonged illumination and modified the light-dependent decrease in sensitivity, which in control conditions approximates to a Weber-Fechner function. The effects of the pump inhibitor on the adaptive properties of rods are associated with an increased time constant of the membrane current attributed to the operation of the Na+:Ca2+,K+ exchanger. The effects of rapid application of the pump inhibitor on the current re-activation are consistent with the idea that significant changes in the internal sodium occur in rods of mammals during background illumination and that they play an important role in the process of light adaptation.

Prenatal monocular enucleation induces a selective loss of low-spatial-frequency cortical responses to the remaining eye.

During early development, interactions between the two eyes are critical in the formation of eye-specific domains within the lateral geniculate nucleus and the visual cortex. When monocular enucleation is done early in prenatal life, it induces remarkable anatomical and functional reorganizations of the visual pathways. Behavioral data have shown a loss in sensitivity to low-spatial-frequency gratings in cats. To correlate the behavioral observations with a possible change in the analysis of contrast at the level of primary visual areas we recorded visual evoked potentials at the 17/18 border in two cats enucleated prenatally (gestational age at enucleation, 39-42 days), three neonatal, two control animals, and one animal with a surgical removal of Y-ganglion fibers. Our results show a strong attenuation in the amplitude of response at all contrast values for gratings of low spatial frequency in prenatally enucleated cats, whereas neonatally enucleated and control animals present responses of comparable amplitude. We conclude that the behavioral results reflect the reduced sensitivity for low frequencies of visual cortical neurons. In addition, we define a critical period for the development of the contrast-sensitivity function that seems to be limited to the prenatal gestation period. We suggest that the prenatal interruption of binocular interactions leads to a functional elimination of the Y-ganglion system.