Dynamic full-field optical coherence tomography module adapted to commercial microscopes allows longitudinal in vitro cell culture study.

Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening.

Adaptive Optics Flood Illumination Ophthalmoscopy in Nonhuman Primates: Findings in Normal and Short-term Induced Detached Retinae.

Objective: To describe adaptive optics flood illumination ophthalmoscopy (AO-FIO) of the photoreceptor layer in normal nonhuman primates (NHPs) and in the case of a short-term induced retinal detachment (RD). Design: Longitudinal fundamental research study. Subjects: Four NHPs were used to image normal retinae with AO-FIO (in comparison with 4 healthy humans); 2 NHPs were used to assess the effects of RD. Intervention: The photoreceptor layer (cone mosaic metrics, including cone density, cone spacing, and cone regularity) was followed with AO-FIO imaging (rtx1, Imagine Eyes) during a surgically induced RD in 2 NHPs using a vehicle solution containing dimethyl sulfoxide, classically used as a chemical solvent. We also performed functional testing of the retina (full-field and multifocal electroretinogram [ERG]). Main Outcome Measures: Correlation of cone mosaic metrics (cone density, spacing, and regularity) between normal retinae of NHPs and humans, and cone metrics, power spectrum, and ERG wave amplitudes after RD. Results: Imaging features were very similar in terms of cone reflectivity, cell density, regularity, and spacing values, showing strong positive correlations between NHPs and humans. After RD, AO-FIO revealed several alterations of the cone mosaic slowly recovering during the 3 months after the reattachment, which were not detected functionally by ERG. Conclusions: These results demonstrate by in vivo AO-FIO imaging the transient structural changes of photoreceptors after an RD in the primate retina. They also provide an interesting illustration of the AO-FIO potential for investigating photoreceptor toxicity during preclinical studies in NHPs with a high translatability to human studies. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker.

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the "don't eat me" signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47-/--mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 1015 photons.cm-2.s-1. Vector doses of 5 × 1010 and 5 × 1011 vg/eye transfected up to 7000 RGCs/mm2 in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.

Phototoxic damage to cone photoreceptors can be independent of the visual pigment: the porphyrin hypothesis.

Lighting is rapidly changing with the introduction of light-emitting diodes (LEDs) in our homes, workplaces, and cities. This evolution of our optical landscape raises major concerns regarding phototoxicity to the retina since light exposure is an identified risk factor for the development of age-related macular degeneration (AMD). In this disease, cone photoreceptors degenerate while the retinal pigment epithelium (RPE) is accumulating lipofuscin containing phototoxic compounds such as A2E. Therefore, it remains unclear if the light-elicited degenerative process is initiated in cones or in the RPE. Using purified cone photoreceptors from pig retina, we here investigated the effect of light on cone survival from 390 to 510 nm in 10 nm steps, plus the 630 nm band. If at a given intensity (0.2 mW/cm²), the most toxic wavelengths are comprised in the visible-to-near-UV range, they shift to blue-violet light (425-445 nm) when exposing cells to a solar source filtered by the eye optics. In contrast to previous rodent studies, this cone photoreceptor phototoxicity is not related to light absorption by the visual pigment. Despite bright flavin autofluorescence of cone inner segment, excitation-emission matrix of this inner segment suggested that cone phototoxicity was instead caused by porphyrin. Toxic light intensities were lower than those previously defined for A2E-loaded RPE cells indicating cones are the first cells at risk for a direct light insult. These results are essential to normative regulations of new lighting but also for the prevention of human retinal pathologies since toxic solar light intensities are encountered even at high latitudes.

VEGF is an autocrine/paracrine neuroprotective factor for injured retinal ganglion neurons.

Vascular endothelial growth factor-A (VEGF) is the angiogenic factor promoting the pathological neovascularization in age-related macular degeneration (AMD) or diabetic macular edema (DME). Evidences have suggested a neurotrophic and neuroprotective role of VEGF, albeit in retina, cellular mechanisms underlying the VEGF neuroprotection remain elusive. Using purified adult retinal ganglion cells (RGCs) in culture, we demonstrated here that VEGF is released by RGCs themselves to promote their own survival, while VEGF neutralization by specific antibodies or traps drastically reduced the RGC survival. These results indicate an autocrine VEGF neuroprotection on RGCs. In parallel, VEGF produced by mixed retinal cells or by mesenchymal stem cells exerted a paracrine neuroprotection on RGCs. Such neuroprotective effect was obtained using the recombinant VEGF-B, suggesting the involvement of VEGF-R1 pathway in VEGF-elicited RGC survival. Finally, glaucomatous patients injected with VEGF traps (ranibizumab or aflibercept) due to either AMD or DME comorbidity, showed a significant reduction of RGC axon fiber layer thickness, consistent with the plausible reduction of the VEGF autocrine stimulation of RGCs. Our results provide evidence of the autocrine neuroprotective function of VEGF on RGCs is crucially involved to preserve injured RGCs such as in glaucomatous patients.

IL-1ß induces rod degeneration through the disruption of retinal glutamate homeostasis.

BACKGROUND: Age-related macular degeneration is characterized by the accumulation of subretinal macrophages and the degeneration of cones, but mainly of rods. We have previously shown that Mononuclear Phagocytes-derived IL-1ß induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1ß but the mechanism is unknown. METHODS: Retinal explants were culture in the presence of human monocytes or IL-1ß and photoreceptor cell survival was analyzed by TUNEL labeling. Glutamate concentration and transcription levels of gene involved in the homeostasis of glutamate were analyzed in cell fractions of explant cultured or not in the presence of IL-1ß. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-ß or monocytes. RESULTS: We here show that IL-1ß does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1ß leads to glutamate-induced rod photoreceptor cell death as it increases the extracellular glutamate concentrations in the retina through the inhibition of its conversion to glutamine in Müller cells, increased release from Müller cells, and diminished reuptake. The inhibition of non-NMDA receptors completely and efficiently prevented rod apoptosis in retinal explants cultured in the presence of IL-1ß or, more importantly, in vivo, in a model of subretinal inflammation. CONCLUSIONS: Our study emphasizes the importance of inflammation in the deregulation of glutamate homeostasis and provides a comprehensive mechanism of action for IL-1ß-induced rod degeneration.

Tissue engineering of retina through high resolution 3-dimensional inkjet bioprinting.

The mammalian retina contains multiple cellular layers, each carrying out a specific task. Such a controlled organization should be considered as a crucial factor for designing retinal therapies. The maintenance of retinal layered complexity through the use of scaffold-free techniques has recently emerged as a promising approach for clinical ocular tissue engineering. In an attempt to fabricate such layered retinal model, we are proposing herein a unique inkjet bioprinting system applied to the deposition of a photoreceptor cells (PRs) layer on top of a bioprinted retinal pigment epithelium (RPE), in a precise arrangement and without any carrier material. The results showed that, after bioprinting, both RPE and PRs were well positioned in a layered structure and expressed their structural markers, which was further demonstrated by ZO1, MITF, rhodopsin, opsin B, opsin R/G and PNA immunostaining, three days after bioprinting. We also showed that considerable amounts of human vascular endothelial growth factors (hVEGF) were released from the RPE printed layer, which confirmed the formation of a functional RPE monolayer after bioprinting. Microstructures of bioprinted cells as well as phagocytosis of photoreceptor outer segments by apical RPE microvilli were finally established through transmission electron microscopy (TEM) imaging. In summary, using this carrier-free bioprinting method, it was possible to develop a reasonable in vitro retina model for studying some sight-threatening diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).

Behavioural responses to a photovoltaic subretinal prosthesis implanted in non-human primates.

Retinal dystrophies and age-related macular degeneration related to photoreceptor degeneration can cause blindness. In blind patients, although the electrical activation of the residual retinal circuit can provide useful artificial visual perception, the resolutions of current retinal prostheses have been limited either by large electrodes or small numbers of pixels. Here we report the evaluation, in three awake non-human primates, of a previously reported near-infrared-light-sensitive photovoltaic subretinal prosthesis. We show that multipixel stimulation of the prosthesis within radiation safety limits enabled eye tracking in the animals, that they responded to stimulations directed at the implant with repeated saccades and that the implant-induced responses were present two years after device implantation. Our findings pave the way for the clinical evaluation of the prosthesis in patients affected by dry atrophic age-related macular degeneration.

Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation.

BACKGROUND: The retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro. METHODS: Primary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed. RESULTS: Chronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and transepithelial resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and transepithelial-resistance changes were prevented by concomitant Transforming Growth Factor ß inhibition. CONCLUSIONS: Our results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.

Noninvasive gene delivery to foveal cones for vision restoration.

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.

Toward smart design of retinal drug carriers: a novel bovine retinal explant model to study the barrier role of the vitreoretinal interface.

Retinal gene delivery via intravitreal injection is hampered by various physiological barriers present in the eye of which the vitreoretinal (VR) interface represents the most serious hurdle. In this study, we present a retinal explant model especially designed to study the role of this interface as a barrier for the penetration of vectors into the retina. In contrast to all existing explant models, the developed model is bovine-derived and more importantly, keeps the vitreous attached to the retina at all times to guarantee an intact VR interface. After ex vivo intravitreal injection into the living retinal explant, the route of fluorescent carriers across the VR interface can be tracked. By applying two different imaging methods on this model, we discovered that the transfer through the VR barrier is size-dependent since 40 nm polystyrene particles are more easily taken up in the retina than 100 and 200 nm sized particles. In addition, we found that removing the vitreous, as commonly done for culture of conventional explants, leads to an overestimation of particle uptake, and conclude that the ultimate barrier to overcome for retinal uptake is undoubtedly the inner limiting membrane. Damaging this matrix resulted in a massive increase in particle transfer into the retina. In conclusion, we have developed a highly relevant ex vivo model that maximally mimics the human in vivo physiology which can be applied as a representative test set-up to assess the potential of promising drug delivery carriers to cross the VR interface.

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show that a red-shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV-ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV- and lentivirus-mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

Subretinal mononuclear phagocytes induce cone segment loss via IL-1ß.

Photo-transduction in cone segments (CS) is crucial for high acuity daytime vision. For ill-defined reasons, CS degenerate in retinitis pigmentosa (RP) and in the transitional zone (TZ) of atrophic zones (AZ), which characterize geographic atrophy (GA). Our experiments confirm the loss of cone segments (CS) in the TZ of patients with GA and show their association with subretinal CD14(+)mononuclear phagocyte (MP) infiltration that is also reported in RP. Using human and mouse MPs in vitro and inflammation-prone Cx3cr1(GFP/GFP) mice in vivo, we demonstrate that MP-derived IL-1ß leads to severe CS degeneration. Our results strongly suggest that subretinal MP accumulation participates in the observed pathological photoreceptor changes in these diseases. Inhibiting subretinal MP accumulation or Il-1ß might protect the CS and help preserve high acuity daytime vision in conditions characterized by subretinal inflammation, such as AMD and RP.

Vibratome sectioning mouse retina to prepare photoreceptor cultures.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer's solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10(5) cells/cm(2). The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Cell specific electrodes for neuronal network reconstruction and monitoring.

Direct interfacing of neurons with electronic devices has been investigated for both prosthetic and neuro-computing applications. In vitro neuronal networks provide great tools not only for improving neuroprostheses but also to take advantage of their computing abilities. However, it is often difficult to organize neuronal networks according to specific cell distributions. Our aim was to develop a cell-type specific immobilization of neurons on individual electrodes to produce organized in vitro neuronal networks on multi-electrode arrays (MEAs). We demonstrate the selective capture of retinal neurons on antibody functionalized surfaces following the formation of self-assembled monolayers from protein-thiol conjugates by simple contact and protein-polypyrrole deposits by electrochemical functionalization. This neuronal selection was achieved on gold for either cone photoreceptors or retinal ganglion neurons using a PNA lectin or a Thy1 antibody, respectively. Anti-fouling of un-functionalized gold surfaces was optimized to increase the capture efficiencies. The technique was extended to electrode arrays by addressing electropolymerization of pyrrole monomers and pyrrole-protein conjugates to active electrodes. Retinal ganglion cell recording on the array further demonstrated the integrity of these neurons following their selection on polypyrrole-coated electrodes. Therefore, this protein-polypyrrole electrodeposition could provide a new approach to generate organized in vitro neuronal networks.

Distinctive glial and neuronal interfacing on nanocrystalline diamond.

Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth.

Taurine is a crucial factor to preserve retinal ganglion cell survival.

Retinal ganglion cells (RGCs) are spiking neurons, which send visual information to the brain, through the optic nerve. RGC degeneration occurs in retinal diseases, either as a primary process or secondary to photoreceptor loss. Mechanisms involved in this neuronal degeneration are still unclear and no drugs directly targeting RGC neuroprotection are yet available. Here, we show that taurine is one factor involved in preserving the RGC survival. Indeed, a taurine depletion induced by the antiepileptic drug, vigabatrin, was incriminated in its retinal toxicity leading to the RGC loss. Similarly, we showed that RGC degeneration can be induced by pharmacologically blocking the taurine-transporter with the chronic administration of a selective inhibitor, which results in a decrease in the taurine levels both in the plasma and in the retinal tissue. Finally, we found that taurine can directly prevent RGC degeneration, occurring either in serum-deprived pure RGC cultures or in animal models presenting an RGC loss (glaucomatous rats and the P23H rats, a model for retinitis pigmentosa). These data suggest that the retinal taurine level is a crucial marker to prevent RGC damage in major retinal diseases.

Taurine provides neuroprotection against retinal ganglion cell degeneration.

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.