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.

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.

Binocular interaction in strabismic kittens deprived of Vision.

Artificial strabismus in kittens decreases the proportion of binocularly driven units in area 17 of the cortex. This change in the binocular interaction of cortical cells also takes place if the kittens are deprived of vision from the day in which the strabismus is surgically produced to the day of the electrophysiological recording. Thus, altered motility of the eyes per se is sufficient to affect binocular interaction in the neurons of area 17 of the cortex.

Variations of the visual responses of the superior colliculus in relation to body roll.

A large percentage of the directional units of the superior colliculus of the curarized cat modify their response to a particular moving visual stimulus as a function of the position of rotation of the animal about its longitudinal axis.

Neural correlate of perceptual adaptation to gratings.

Exposure of simple cells of the cat striate cortex to high-contrast drifting gratings greatly reduces the subsequent response of the cells to low-contrast gratings. This adaptation effect has an average duration of 30 seconds and shows interocular transfer and selectivity for spatial frequency and orientation. This effect is strikingly similar to the perceptual adaptation to high-contrast gratings.

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.

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.

Two systems of branching axons in monkey's retina.

Several monkey retinae were stained, by using the reduced silver technique, in order to analyse long-distance intraretinal connections. Long, bifurcating processes covering very large areas were identified. Morphological investigation of these processes suggest that they are members of two different systems of branching axons. The first population of these processes originates as axon collaterals from cell in the ganglion cell layer. These cells have a relatively large, elongated soma and straight, sparsely branching dendrites, stratified in the vitreal half of the inner plexiform layer. The main axon (0.6 microns average diameter) passes along the optic fibre bundles, disappearing into the optic disk, whilst its collaterals run mainly in the inner plexiform layer. A cell showing similar morphology has also been found in the ganglion cell layer of a cat retina. The second population of processes consists of very thick fibres (2.1 microns average diameter) apparently originating from the optic disk. The main branches run in the space between the optic fibre layer and the ganglion cell layer, with short, secondary processes crossing the ganglion cel layer orthogonally. Many higher-order processes originate from the second-order branches; these run almost horizontally in the inner plexiform layer. The ganglion cells generating axon collaterals may constitute an intraretinal firing synchronization system, or they may be a residual feature of retinal development. The centrifugal fibres may be related to the sensitivity control during retinal dark adaptation.

Interplay between the dendritic trees of alpha and beta ganglion cells during the development of the cat retina.

We have analyzed the effect of a small lesion to the retina of a two-day-old kitten and observed that after degeneration of ganglion cells whose axons were severed, a restricted region of the retina remained depleted of cells. Cells located near the borders of the depleted zone showed an abnormal elongation of dendrites towards the bare area. By means of a computer-aided system, we analyzed the whole population of cells at the two borders, and in agreement with previous data found that the effect was most prominent at the border and progressively decreased to eventually disappear at a distance of approximately 500 microns. The distance from the border, however, is not the only factor to influence the degree of asymmetry; with comparable distances, the vicinity of an alpha-cell reduces the projection of the beta-cell dendrites toward the empty area. We suggest that the organization of the adult retinal pattern is also influenced by interactions occurring between dendrites of different classes of ganglion cells.

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.

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.

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.

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.

Monocular deprivation in kittens differently affects crossed and uncrossed visual pathways.

The effects of monocular deprivation (MD) on the crossed and uncrossed visual projections were studied using both electrophysiological and behavioural criteria. Our results show that Visual Evoked Potentials (VEPs) from the deprived eye (DE) in response to contrast reversing gratings are more reduced in the ipsilateral than in the contralateral cortex. This suggests a different sensitivity of the crossed and uncrossed visual pathways to MD. In the behavioural experiments comparable findings were obtained.

The visual acuity of the lynx.

Visual evoked potentials were recorded from the occipital scalp of two anaesthetized Lynx (Lynx europea) in response to alternating gratings of various spatial frequencies and contrasts. The visual acuity of the Lynx was found to be around 5-6 c/deg, i.e. very close to the visual acuity of the cat and by far inferior to human acuity.

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.

The pattern electroretinogram in response to colour contrast in man and monkey.

Steady-state pattern-reversal electroretinograms (PERG) were recorded from both monkeys and humans in response to tartan patterns modulated in both space and time in either luminance contrast or chromatic contrast. In both species, all types of patterns cause a strong modulation of the second-harmonic of the PERG. There was no measurable dependency of the PERG on the colour of the stimulus per se: both in humans and monkeys, stimuli with green-black, red-black or yellow-black modulation of the same mean luminance and of the same contrast, produced identical results. However, chromatic stimuli with modulation between equiluminant red and green produced a qualitatively different PERG: the amplitude was lower, particularly at high temporal frequencies, and there was a clear phase lag corresponding to a difference in processing time of about 20 ms.

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.