Silvia Bisti

A polymer optoelectronic interface restores light sensitivity in blind rat retinas

Interfacing organic electronics with biological substrates offers new possibilities for biotechnology due to the beneficial properties exhibited by organic conducting polymers. These polymers have been used for cellular interfaces in several fashions, including cellular scaffolds, neural probes, biosensors and actuators for drug release. Recently, an organic photovoltaic blend has been exploited for neuronal stimulation via a photo-excitation process. Here, we document the use of a single-component organic film of poly(3-hexylthiophene) (P3HT) to trigger neuronal firing upon illumination. Moreover, we demonstrate that this bio-organic interface restored light sensitivity in explants of rat retinas with light-induced photoreceptor degeneration. These findings suggest that all-organic devices may play an important future role in sub-retinal prosthetic implants.

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.

Saffron Supplement Maintains Morphology and Function after Exposure to Damaging Light in Mammalian Retina

PURPOSE To test whether the saffron extract (Crocus sativus L.) given as a dietary supplement counteracts the effects of continuous light exposure in the albino rat retina. METHODS Three experimental groups of Sprague-Dawley rats were used. Experimental animals were prefed either saffron or beta-carotene (1 mg extract/kg/d) before they were exposed to bright continuous light (BCL) for 24 hours. Flash electroretinograms (fERGs) were recorded in control and treated rats the day before and 1 week after light exposure. At the end of the second recording session, the animals were killed and the retinas were quickly removed, fixed, cryosectioned, and labeled so that the thickness of the outer nuclear layer (ONL) could be analyzed. Changes in protein level and cellular localization of fibroblast growth factor (FGF)2 were determined by Western blot analysis and retinal immunohistochemistry, respectively. In a second series of experiments, rats were killed at the end of light exposure, and the amount of apoptotic figures in the ONL was assessed by terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). BCL induced DNA fragmentation, characteristic of dying cells, almost exclusively in the photoreceptor layer. The rate of photoreceptor death induced by BCL is expressed as the frequency of TUNEL-positive profiles per millimeter. RESULTS The photoreceptor layer was largely preserved in saffron-treated animals because it was the fERG response. In addition, the rate of photoreceptor death induced by BCL appeared drastically reduced in treated animals. In beta-carotene prefeeding experiments, morphologic analysis showed preservation of the ONL similar to that obtained with saffron prefeeding, whereas the fERG response was unrecordable. Western blot analysis showed that exposure to light induced a strong upregulation of FGF2 in control and beta-carotene-treated rats, but s no change was noted in saffron-treated rats. CONCLUSIONS These results show that saffron may protect photoreceptors from retinal stress, maintaining both morphology and function and probably acting as a regulator of programmed cell death.

Pattern-reversal electroretinogram in response to chromatic stimuli: I. Humans.

We have studied the steady-state PERG in human subjects in response to red-green plaid patterns modulated either in luminance or in chromaticity or both. By varying the relative luminance of the red and green components, a value could be obtained at which the PERG amplitude was either minimum or locally maximum. This always occurred at equiluminance, as measured by standard psychophysical techniques. PERG amplitude and phase were measured as a function of spatial and temporal frequency of sinusoidal contrast reversal. In both space and time, the response to chromatic patterns was low-pass, while that to luminance was band-pass, and extended to higher spatial and temporal frequencies. The phase of the PERG to chromatic stimuli was systematically lagged compared with that to luminance stimuli, by an amount corresponding to about 20 ms under our experimental conditions. The variation of phase with temporal frequency suggested an apparent latency of about 67 ms for color contrast compared with 47 ms for luminance. These estimates were confirmed with separate measurements of transient PERGs to abrupt contrast reversal. For both luminance and chromatic stimuli, the amplitude of PERGs increases with increasing stimulus contrast. By summing vectorially the responses to appropriate luminance and chromatic contrasts, we were able to predict with accuracy the response as a function of color ratio (ratio of red to total luminance). The above findings all agree with those reported in the accompanying paper for the monkey PERG (Morrone et al., 1994), and indicate that the differences in response latency and integration time of luminance and chromatic stimuli observed by psychophysical and VEP techniques may arise at least in part from the properties of retinal mechanisms.

Sensitivity to spatial frequency and contrast of visual cells in the cat superior colliculus.

Abstract The dependence of the activity of single cells in the superficial layers of the superior colliculus of the cat upon the spatial frequency and contrast of a moving sinusoidal grating was analysed quantitatively. The responses of about 65% of the units tested were found to be spatial-frequency dependent. The range of sensitivity was relatively broad and somewhat similar to that observed in the complex cells of the visual cortex. No responses of collicular units were found above 2 c/deg. The spatial-frequency sensitive units also showed a marked sensitivity to variations of grating contrast. There appeared to be no simple correlation between receptive field characteristics and cell sensitivity to either spatial frequency or contrast. Chronic ablation of the cortical areas known to project to the superior colliculus (areas 17, 18, 19 and Clare-Bishop) did not abolish the sensitivity of collicular units to spatial periodical stimuli. The findings suggest the existence of some internal organization of collicular units, able to perform an analysis, albeit a primitive one, of spatial information.

Location of CNTFRα on outer segments: evidence of the site of action of CNTF in rat retina

Ciliary neurotrophic factor (CNTF) is an important factor in the retinas mechanisms of self-protection. It is generated by retinal glial cells in response to stress, and has a significant protective effect on retinal neurones. In this study we have identified the location of the alpha component of the CNTF receptor complex (CNTFRalpha) in rat retina, using immunohistochemistry and high-resolution confocal microscopy. The major location of CNTFRalpha is on photoreceptor outer segments. More scattered, granular forms of CNTFRalpha were identified in association with Müller cell processes in other retinal layers. Colocalisation of CNTF with CNTFRalpha, suggestive of ligand-receptor binding, was detected on outer segments, and in both normal retinas and retinas stressed by light or oxygen. Results provide evidence that the principal site of CNTF action is the outer segments of photoreceptors. This confirms the known ability of CNTF to protect photoreceptors against stress, and suggest that it acts by modulating mechanisms specific to the outer segment, such as the phototransduction cascade or the membrane channels, which control dark current.

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.

Permanent Functional Reorganization of Retinal Circuits Induced by Early Long-Term Visual Deprivation

Early sensory experience shapes the functional and anatomical connectivity of neuronal networks. Light deprivation alters synaptic transmission and modifies light response properties in the visual system, from retinal circuits to higher visual centers. These effects are more pronounced during a critical period in juvenile life and are mostly reversed by restoring normal light conditions. Here we show that complete light deprivation, from birth to periods beyond the critical period, permanently modifies the receptive field properties of retinal ganglion cells. Visual deprivation reduced both the strength of light responses in ganglion cells and their receptive field size. Light deprivation produced an imbalance in the ratio of inhibitory to excitatory inputs, with a shift toward larger inhibitory conductances. Ganglion cell receptive fields in visually deprived animals showed a spatial mismatch of inhibitory and excitatory inputs and inhibitory inputs were highly scattered over the receptive field. These results indicate that visual experience early in life is critical for the refinement of retinal circuits and for appropriate signaling of the spatiotemporal properties of visual stimuli, thus influencing the response properties of neurons in higher visual centers and their processing of visual information.

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.

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.