Gradual Increase in Environmental Light Intensity Induces Oxidative Stress and Inflammation and Accelerates Retinal Neurodegeneration.

Purpose: Retinitis pigmentosa (RP) is a blinding neurodegenerative disease of the retina that can be affected by many factors. The present study aimed to analyze the effect of different environmental light intensities in rd10 mice retina. Methods: C57BL/6J and rd10 mice were bred and housed under three different environmental light intensities: scotopic (5 lux), mesopic (50 lux), and photopic (300 lux). Visual function was studied using electroretinography and optomotor testing. The structural and morphological integrity of the retinas was evaluated by optical coherence tomography imaging and immunohistochemistry. Additionally, inflammatory processes and oxidative stress markers were analyzed by flow cytometry and western blotting. Results: When the environmental light intensity was higher, retinal function decreased in rd10 mice and was accompanied by light-dependent photoreceptor loss, followed by morphological alterations, and synaptic connectivity loss. Moreover, light-dependent retinal degeneration was accompanied by an increased number of inflammatory cells, which became more activated and phagocytic, and by an exacerbated reactive gliosis. Furthermore, light-dependent increment in oxidative stress markers in rd10 mice retina pointed to a possible mechanism for light-induced photoreceptor degeneration. Conclusions: An increase in rd10 mice housing light intensity accelerates retinal degeneration, activating cell death, oxidative stress pathways, and inflammatory cells. Lighting intensity is a key factor in the progression of retinal degeneration, and standardized lighting conditions are advisable for proper analysis and interpretation of experimental results from RP animal models, and specifically from rd10 mice. Also, it can be hypothesized that light protection could be an option to slow down retinal degeneration in some cases of RP.

New Nrf2-Inducer Compound ITH12674 Slows the Progression of Retinitis Pigmentosa in the Mouse Model rd10.

BACKGROUND/AIMS: It is well established that oxidative stress and inflammation are common pathogenic features of retinal degenerative diseases. ITH12674 is a novel compound that induces the transcription factor Nrf2; in so doing, the molecule exhibits anti-inflammatory, and antioxidant properties, and affords neuroprotection in rat cortical neurons subjected to oxidative stress. We here tested the hypothesis that ITH12674 could slow the retinal degeneration that causes blindness in rd10 mice, a model of retinitis pigmentosa. METHODS: Animals were intraperitoneally treated with 1 or 10 mg/Kg ITH12674 or placebo from P16 to P30. At P30, retinal functionality and visual acuity were analyzed by electroretinography and optomotor test. By immunohistochemistry we quantified the photoreceptor rows and analyzed their morphology and connectivity. Oxidative stress and inflammatory state was studied by Western blot, and microglia reactivity was monitored by flow cytometry. The blood-brain barrier permeation of ITH12674 was evaluated using a PAMPA-BBB assay. RESULTS: In rd10 mice treated with 10 mg/Kg of the compound, the following changes were observed (with respect to placebo): (i) a decrease of vision loss with higher scotopic a- and b-waves; (ii) increased visual acuity; (iii) preservation of cone photoreceptors morphology, as well as their synaptic connectivity; (iv) reduced expression of TNF-α and NF-κB; (v) increased expression of p38 MAPK and Atg12-Atg5 complex; and (vi) decreased CD11c, MHC class II and CD169 positive cell populations. CONCLUSION: These data support the view that a Nrf2 inducer compound may arise as a new therapeutic strategy to combat retinal neurodegeneration. At present, we are chemically optimising compound ITH12674 with the focus on improving its neuroprotective potential in retinal neurodegenerative diseases.

The Absence of Toll-Like Receptor 4 Mildly Affects the Structure and Function in the Adult Mouse Retina.

The innate immune Toll-like receptor (TLR) family plays essential roles in cell proliferation, survival and function of the central nervous system. However, the way in which TLRs contribute to the development and maintenance of proper retinal structure and function remains uncertain. In this work, we assess the effect of genetic TLR4 deletion on the morphology and function of the retina in mice. Visual acuity and retinal responsiveness were evaluated in TLR4 knockout and wild type C57BL/6J control mice by means of an optomotor test and electroretinography, respectively, from P20 to P360. Retinal structure was also analyzed in both strains using confocal and electron microscopy. ERG data showed impaired retinal responsiveness in TLR4 KO mice, in comparison to wild type animals. The amplitudes of the scotopic a-waves were less pronounced in TLR4-deficient mice than in wild-type animals from P30 to P360, and TLR4 KO mice presented scotopic b-wave amplitudes smaller than those of age-matched control mice at all ages studied (P20 to P360). Visual acuity was also relatively poorer in TLR4 KO as compared to C57BL/6J mice from P20 to P360, with significant differences at P30 and P60. Immunohistochemical analysis of retinal vertical sections showed no differences between TLR4 KO and C57BL/6J mice, in terms of either photoreceptor number or photoreceptor structure. Horizontal cells also demonstrated no morphological differences between TLR4 KO and wild-type mice. However, TLR4 KO mice exhibited a lower density of bipolar cells (15% less at P30) and thus fewer bipolar cell dendrites than the wild type control mouse, even though both confocal and electron microscopy images showed no morphologic abnormalities in the synaptic contacts between the photoreceptors and second order neurons. Microglial cell density was significantly lower (26% less at P30) in TLR4 KO mice as compared to wild-type control mice. These results suggest that TLR4 deletion causes functional alterations in terms of visual response and acuity, probably through the loss of bipolar cells and microglia, but this receptor is not essential for the processing of visual information in the retina.

Effect of magnetically simulated zero-gravity and enhanced gravity on the walk of the common fruitfly.

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly (Drosophila melanogaster) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earth's surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0g*) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2g*) and a normal gravity environment (1g*) within the spatially varying field. The flies had a larger mean speed in 0g* than in 1g*, and smaller in 2g*. The mean square distance travelled by the flies grew more rapidly with time in 0g* than in 1g*, and slower in 2g*. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.

Rotenone induces degeneration of photoreceptors and impairs the dopaminergic system in the rat retina.

Rotenone is a widely used pesticide and a potent inhibitor of mitochondrial complex I (NADH-quinone reductase) that elicits the degeneration of dopaminergic neurons and thereby the appearance of a parkinsonian syndrome. Here we have addressed the alterations induced by rotenone at the functional, morphological and molecular levels in the retina, including those involving both dopaminergic and non-dopaminergic retinal neurons. Rotenone-treated rats showed abnormalities in equilibrium, postural instability and involuntary movements. In their outer retina we observed a loss of photoreceptors, and a reduced synaptic connectivity between those remaining and their postsynaptic neurons. A dramatic loss of mitochondria was observed in the inner segments, as well as in the axon terminals of photoreceptors. In the inner retina we observed a decrease in the expression of dopaminergic cell molecular markers, including loss of tyrosine hydroxylase immunoreactivity, associated with a reduction of the dopaminergic plexus and cell bodies. An increase in immunoreactivity of AII amacrine cells for parvalbumin, a Ca(2+)-scavenging protein, was also detected. These abnormalities were accompanied by a decrease in the amplitude of scotopic and photopic a- and b-waves and an increase in the b-wave implicit time, as well as by a lower amplitude and greater latency in oscillatory potentials. These results indicate that rotenone induces loss of vision by promoting photoreceptor cell death and impairment of the dopaminergic retinal system.

Nicotinic acetylcholine receptor properties are modulated by surrounding lipids: an in vivo study.

In vitro studies carried out on liposomes of defined composition showed that nicotinic acetylcholine receptors (nAChRs) are fully functional when they are reconstituted in a heterogeneous lipid matrix, such as that provided by crude soybean (asolectin [R-Aso]) lipids. However, when they are reconstituted in plain phosphatidylcholine (R-PC) lipids, their functional activity is completely lost (Fong and McNamee, 1986). This kind of study also pointed out that phosphatidic acid (PA) and cholesterol (Chol) play an important role in preserving the ability of this protein to exhibit an optimal channel activity (Fong and McNamee, 1986). Furthermore, it has been shown recently that nAChR, itself, induces the formation of specific PA-rich lipid domains (Poveda et al., 2002). Because Xenopus oocytes incorporate functionally into their plasma membrane nAChRs after intracellular injection of liposomes bearing this protein (Morales et al., 1995), the aim of this work was to determine the effect of the reconstitution lipid matrix on the functional properties of the transplanted nAChRs.

Morphological impairments in retinal neurons of the scotopic visual pathway in a monkey model of Parkinson's disease.

Physiological abnormalities resulting from death of dopaminergic neurons of the central nervous system in Parkinson's disease also extend to the retina, resulting in impaired visual functions. In both parkinsonian patients and animal models, low levels of dopamine and loss of dopaminergic cells in the retina have been reported. However, the morphology and connectivity of their postsynaptic neurons, the amacrine cells, have not been analyzed. Here we report, with macaques chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of Parkinson's disease, that morphological impairments in dopaminergic retinal neurons and their plexus in the inner retina are accompanied by an immunoreactivity decrease in gamma-aminobutyric acidergic and glycinergic amacrine cells. Especially deteriorated were AII amacrine cells, the main neuronal subtype postsynaptic to dopaminergic cells, which exhibited a marked loss of lobular appendages and dendritic processes. Concomitantly, electrical synapses among AII cells, as well as chemical synapses between these and rod bipolar cells, were highly deteriorated in parkinsonian monkeys. These results highlight that the scotopic visual pathway is severely impaired in the parkinsonian condition and provide a morphological basis for a number of abnormalities found in electrophysiological and psychophysical trials in Parkinson's disease patients and animal models.