Rita Maccarone

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.

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.

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.

Bright light exposure reduces TH-positive dopamine neurons: implications of light pollution in Parkinson's disease epidemiology.

This study explores the effect of continuous exposure to bright light on neuromelanin formation and dopamine neuron survival in the substantia nigra. Twenty-one days after birth, Sprague–Dawley albino rats were divided into groups and raised under different conditions of light exposure. At the end of the irradiation period, rats were sacrificed and assayed for neuromelanin formation and number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. The rats exposed to bright light for 20 days or 90 days showed a relatively greater number of neuromelanin-positive neurons. Surprisingly, TH-positive neurons decreased progressively in the substantia nigra reaching a significant 29% reduction after 90 days of continuous bright light exposure. This decrease was paralleled by a diminution of dopamine and its metabolite in the striatum. Remarkably, in preliminary analysis that accounted for population density, the age and race adjusted Parkinsons disease prevalence significantly correlated with average satellite-observed sky light pollution.

Characterization of a Polymer-Based, Fully Organic Prosthesis for Implantation into the Subretinal Space of the Rat

Replacement strategies arise as promising approaches in case of inherited retinal dystrophies leading to blindness. A fully organic retinal prosthesis made of conjugated polymers layered onto a silk fibroin substrate is engineered. First, the biophysical and surface properties are characterized; then, the long-term biocompatibility is assessed after implantation of the organic device in the subretinal space of 3-months-old rats for a period of five months. The results indicate a good stability of the subretinal implants over time, with preservation of the physical properties of the polymeric layer and a tight contact with the outer retina. Immunoinflammatory markers detect only a modest tissue reaction to the surgical insult and the foreign body that peaks shortly after surgery and progressively decreases with time to normal levels at five months after implantation. Importantly, the integrity of the polymeric layer in direct contact with the retinal tissue is preserved after five months of implantation. The recovery of the foreign-body tissue reaction is also associated with a normal b-wave in the electroretinographic response. The results demonstrate that the device implanted in nondystrophic eyes is well tolerated, highly biocompatible, and suitable as retinal prosthesis in case of photoreceptor degeneration.

Saffron and retina: neuroprotection and pharmacokinetics.

Age-related macular degeneration (AMD) is a retinal neurodegenerative disease whose development and progression are the results of a complex interaction between genetic and environmental risk factors. Both oxidative stress and chronic inflammation play a significant role in the pathogenesis of AMD. Experimental studies in rats with light-induced photoreceptors degeneration demonstrated that saffron may protect photoreceptor from retinal stress, preserving both morphology and function and probably acting as a regulator of programmed cell death, in addition to its antioxidant and anti-inflammatory properties. Recently, a randomized clinical trial showed that in patients with early AMD, dietary supplementation with saffron was able to improve significantly the retinal flicker sensitivity suggesting neuroprotective effect of the compound. Here, we examine the progress of saffron dietary supplementation both in animal model and AMD patients, and discuss the potential and safety for using dietary saffron to treat retinal degeneration.

Long-term treatment of the developing retina with the metabotropic glutamate agonist APB induces long-term changes in the stratification of retinal ganglion cell dendrites.

The gradual restriction of initially multistratified retinal ganglion cell (RGC) dendrites into ON and OFF sublaminae of the inner plexiform layer (IPL) can be effectively blocked by treating the developing retina with 2-amino-4-phosphonobutyrate (APB), the metabotropic glutamate agonist, or by light deprivation. Previous studies have focused on the short-term consequences of such manipulations, so the long-term effects of arresting dendritic stratification on the structural development of RGCs are as yet unknown. In the present study, we have addressed this issue by performing a morphological analysis of α RGCs labeled by retrograde transport of horseradish peroxidase injected into the dorsal lateral geniculate nucleus of adult cats that received monocular injections of APB from postnatal (P) day 2 until P30. A large proportion of the α cells in the APB-treated eye (44%) were found to have multistratified dendrites that terminated in both the ON and OFF sublaminae of the IPL. The dendritic arborization pattern in the sublaminae of the IPL of these cells was asymmetric, showing a variety of forms. Immunolabeling of retinal cross-sections showed that mGLUR6 receptors appeared normal in density and location, while qualitative observation suggested an increase in the axonal arborization of rod bipolar cells. These findings indicate that long-term treatment of the neonatal retina with APB induces a long- lasting structural reorganization in retinal circuitry that most likely accounts for some of the previously described changes in the functional properties of RGCs.

Functional effect of Saffron supplementation and risk genotypes in early age-related macular degeneration: a preliminary report.

BackgroundTo determine whether the functional effects of oral supplementation with Saffron, a natural compound that proved to be neuroprotective in early age-related macular degeneration, are influenced by complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) risk genotypes.MethodsThirty-three early AMD patients, screened for CFH (rs1061170) and ARMS2 (rs10490924) polymorphisms and receiving Saffron oral supplementation (20 mg/day) over an average period of treatment of 11 months (range, 6–12), were longitudinally evaluated by clinical examination and focal electroretinogram (fERG)-derived macular (18°) flicker sensitivity estimate. fERG amplitude and macular sensitivity, the reciprocal value of the estimated fERG amplitude threshold, were the main outcome measures.ResultsAfter three months of supplementation, mean fERG amplitude and fERG sensitivity improved significantly when compared to baseline values (p < 0.01). These changes were stable throughout the follow-up period. No significant differences in clinical and fERG improvements were observed across different CFH or ARMS2 genotypes.ConclusionsThe present results indicate that the functional effect of Saffron supplementation in individual AMD patients is not related to the major risk genotypes of disease.

Interleukin 1-beta modulates the effects of hypoxia in neuronal culture

In order to study the role of interleukin-1beta (IL-1beta) in homeostasis, hypoxia and recovery of neuronal cells, we studied the expression and release of tumor necrosis factor-alpha (TNF-alpha) and nerve growth factor (NGF), in relation to the presence or absence of this cytokine in culture medium. Moreover, we evaluated cell mortality in the same conditions. For this aim, we used untreated and IL-1beta pre-immunoneutralized hippocampal neuronal cultures exposed to mild hypoxic stress and left to reoxygenate. Semiquantitative reverse-transciptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) determined gene expression and protein levels. Mild hypoxic stress provokes a decrease in both the expression and release of TNF-alpha and NGF. IL-1beta neutralization results in an inversion of this pattern since treated hypoxic cultures exhibited an increase of both expression and release of NGF. In pretreated hypoxic cells the increased expression of TNF-alpha was not followed by a rise in release. Reoxygenation reversed the observed effects in both cultures and the levels of cytokine expression and release were approaching control values. Our data show that in physiological conditions IL-1beta may have a neuroprotective action through positive modulation of NGF. Contrary to that, in presence of insult, IL-1beta may have an opposite role, since neutralization provoked an increase of expression and release of NGF. In addition, we demonstrated that neuronal cells are biochemically capable, not only of maintaining and recovering the homeostasis, but also of activating the appropriate response to insult. IL-1beta may have a pivotal role in this mechanism through the modulation of NGF and to a lesser degree of TNF-alpha.

Gene expression and protein localization of calmodulin- dependent phosphodiesterase in adult rat retina

Calcium calmodulin‐dependent cyclic nucleotide phosphodiesterase (PDE1) was identified in crude extract and immunolabeled sections of rat retina. Both cAMP and cGMP PDE activities were stimulated by calcium‐calmodulin (4.7‐fold and 2.3‐fold, respectively). To characterize PDE1 isoforms in retinal cells further, we used antibodies that specifically recognize PDE1 gene products. PDE1B antibody stained a band at molecular mass of 63 kDa whereas PDE1C antibody recognized two bands at 74‐ and 70‐kDa molecular masses. Two PDE1A antibodies (against N‐terminal and C‐terminal peptides) detected a band at 79 kDa never described before. Immunohistochemical analysis showed a distribution of PDE1A in the outer retina with a bright fluorescence in the outer segments of photoreceptors. PDE1B is uniformly distributed across the retina. PDE1C is confined mainly to the inner retina, with a precise localization in the inner nuclear layer. Immunostaining with choline acetyltransferase antibody indicates localization in cholinergic amacrine cell. The present data provide evidence of expression of PDE1 isoforms in mammalian retina with a complementary distribution of PDE1A and PDE1C, suggesting different roles in retinal function.