RESUMEN

Retinal degeneration is one of the main causes of visual impairment and blindness. One group of retinal degenerative diseases, leading to the loss of photoreceptors, is collectively termed retinitis pigmentosa. In this group of diseases, the remaining retina is largely spared from initial cell death making retinal ganglion cells an interesting target for vision restoration methods. However, it is unknown how downstream brain areas, in particular the visual cortex, are affected by the progression of blindness. Visual deprivation studies have shown dramatic changes in the electrophysiological properties of visual cortex neurons, but changes on a cellular level in retinitis pigmentosa have not been investigated yet. Therefore, we used the rd10 mouse model to perform patch-clamp recordings of pyramidal neurons in layer 2/3 of the primary visual cortex to screen for potential changes in electrophysiological properties resulting from retinal degeneration. Compared to wild-type C57BL/6 mice, we only found an increase in intrinsic excitability around the time point of maximal retinal degeneration. In addition, we saw an increase in the current amplitude of spontaneous putative inhibitory events after a longer progression of retinal degeneration. However, we did not observe a long-lasting shift in excitability after prolonged retinal degeneration. Together, our results provide evidence of an intact visual cortex with promising potential for future therapeutic strategies to restore vision.

RESUMEN

Müller glia are the principal macroglia of the retina and support retinal neurons both in health and disease. In retinitis pigmentosa (RP), a highly heterogeneous inherited retinal disorder, the most common form of pathology involves primary rod degeneration, followed by secondary cone death. To investigate Müller glia responses to rod degeneration, we performed droplet-based single-cell RNA sequencing in the rd10 mouse model of RP during primary rod degeneration. We confirmed known MG behavior on gliosis, metabolic, and immune functions. Pde6brd10 Müller glia also exhibited an increased expression of histocompatibility complex members, which might arise from a novel immune function of Müller glia in RP. We also describe a possible decrease in glial lipid biogenesis, which might affect degenerating photoreceptors.

Asunto(s)

RESUMEN

BACKGROUND: Retinitis pigmentosa is a rod-cone degenerative disease that induces irreversible vision loss. This study probed the protective capacity of mesenchymal stem cell-derived small EVs (MSC-EVs) on the retinas of rd10 mice and the underlying mechanism. METHODS: MSC-EVs were injected into the vitreous of rd10 mice at postnatal day 14 and P21; morphology and function were examined at P28. The mechanism of action was explored by using co-culture of photoreceptor cell line 661 W and microglia cell line BV2. RESULTS: Treatment with MSC-EVs increased the survival of photoreceptors and preserved their structure. Visual function, as reflected by optomotor and electroretinogram responses, was significantly enhanced in MSC-EVs-treated rd10 mice. Mechanistically, staining for Iba1, GFAP, F4/80, CD68 and CD206 showed that MSC-EVs suppressed the activation of microglial, Müller glial and macrophages. Furthermore, western blotting showed that the treatment inhibited the NF-κB pathway. RNA-seq and qPCR showed that MSC-EVs upregulated anti-inflammatory cytokines while downregulating pro-inflammatory cytokines. MSC-EVs application in vitro decreased the number of TUNEL-positive 661 W cells co-cultured with LPS-stimulated BV2, with similar impact on the cytokine expression as in vivo study. Genetic screening predicted miR-146a to be the downstream target of MSC-EVs, which was detected in MSC-EVs and upregulated in co-cultured 661 W cells and BV2 cells after MSC-EVs treatment. Upregulation of miR-146a by using its mimic decreased the expression of the transcription factor Nr4a3, and its downregulation inhibition promoted Nr4a3 expression in both 661 W and BV2 cells. Nr4a3 was further identified as the target gene of miR-146a by dual-luciferase assay. Furthermore, overexpressing miR-146a significantly decreased the expression of LPS-induced pro-inflammatory cytokines in BV2 cells. CONCLUSIONS: MSC-EVs delays retinal degeneration in rd10 mice mainly by its anti-inflammatory effect via the miR-146a-Nr4a3axis. Hence, MSC-EVs may be used in the treatment of neurodegenerative diseases.

Asunto(s)

RESUMEN

Retinitis pigmentosa is a retinal disease characterized by photoreceptor degeneration. There is currently no effective treatment for retinitis pigmentosa. Although a mixture of lutein and other antioxidant agents has shown promising effects in protecting the retina from degeneration, the role of lutein alone remains unclear. In this study, we administered intragastric lutein to Pde6brd10 model mice, which display degeneration of retinal photoreceptors, on postnatal days 17 (P17) to P25, when rod apoptosis reaches peak. Lutein at the optimal protective dose of 200 mg/kg promoted the survival of photoreceptors compared with vehicle control. Lutein increased rhodopsin expression in rod cells and opsin expression in cone cells, in line with an increased survival rate of photoreceptors. Functionally, lutein improved visual behavior, visual acuity, and retinal electroretinogram responses in Pde6brd10 mice. Mechanistically, lutein reduced the expression of glial fibrillary acidic protein in Müller glial cells. The results of this study confirm the ability of lutein to postpone photoreceptor degeneration by reducing reactive gliosis of Müller cells in the retina and exerting anti-inflammatory effects. This study was approved by the Laboratory Animal Ethics Committee of Jinan University (approval No. LACUC-20181217-02) on December 17, 2018.

RESUMEN

Retinitis pigmentosa (RP) is a family of inherited disorders caused by the progressive degeneration of retinal photoreceptors. There is no cure for RP, but recent research advances have provided promising results from many clinical trials. All these therapeutic strategies are focused on preserving existing photoreceptors or substituting light-responsive elements. Vision recovery, however, strongly relies on the anatomical and functional integrity of the visual system beyond photoreceptors. Although the retinal structure and optic pathway are substantially preserved at least in early stages of RP, studies describing the visual cortex status are missing. Using a well-established mouse model of RP, we analyzed the response of visual cortical circuits to the progressive degeneration of photoreceptors. We demonstrated that the visual cortex goes through a transient and previously undescribed alteration in the local excitation/inhibition balance, with a net shift towards increased intracortical inhibition leading to improved filtering and decoding of corrupted visual inputs. These results suggest a compensatory action of the visual cortex that increases the range of residual visual sensitivity in RP.

Asunto(s)

RESUMEN

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.

Asunto(s)

RESUMEN

Objective. Most neuroprosthetic implants employ pulsatile square-wave electrical stimuli, which are significantly different from physiological inter-neuronal communication. In case of retinal neuroprosthetics, which use a certain type of pulsatile stimuli, reliable object and contrast discrimination by implanted blind patients remained challenging. Here we investigated to what extent simple objects can be discriminated from the output of retinal ganglion cells (RGCs) upon sinusoidal stimulation.Approach. Spatially confined objects were formed by different combinations of 1024 stimulating microelectrodes. The RGC activity in theex vivoretina of photoreceptor-degenerated mouse, of healthy mouse or of primate was recorded simultaneously using an interleaved recording microelectrode array implemented in a CMOS-based chip.Main results. We report that application of sinusoidal electrical stimuli (40 Hz) in epiretinal configuration instantaneously and reliably modulates the RGC activity in spatially confined areas at low stimulation threshold charge densities (40 nC mm-2). Classification of overlapping but spatially displaced objects (1° separation) was achieved by distinct spiking activity of selected RGCs. A classifier (regularized logistic regression) discriminated spatially displaced objects (size: 5.5° or 3.5°) with high accuracy (90% or 62%). Stimulation with low artificial contrast (10%) encoded by different stimulus amplitudes generated RGC activity, which was classified with an accuracy of 80% for large objects (5.5°).Significance. We conclude that time-continuous smooth-wave stimulation provides robust, localized neuronal activation in photoreceptor-degenerated retina, which may enable future artificial vision at high temporal, spatial and contrast resolution.

Asunto(s)

RESUMEN

Sigma 1 Receptor (Sig1R), a pluripotent modulator of cell survival, is a promising target for treatment of retinal degenerative diseases. Previously, we reported that administration of the high-affinity, high-specificity Sig1R ligand (+)-pentazocine, ((+)-PTZ) beginning at post-natal day 14 (P14) and continuing every other day improves visual acuity and delays loss of photoreceptor cells (PRCs) in the Pde6ßrd10/J (rd10) mouse model of retinitis pigmentosa. Whether administration of (+)-PTZ, at time points concomitant with (P18) or following (P21, P24) onset of PRC death, would prove neuroprotective was investigated in this study. Rd10 mice were administered (+)-PTZ intraperitoneally [0.5 mg/kg], starting at either P14, P18, P21 or P24. Injections continued every other day through P42. Visual acuity was assessed using the optokinetic tracking response (OKR). Rd10 mice treated with (+)-PTZ beginning at P14 retained visual acuity for the duration of the study (~0.33 c/d at P21, ~0.38 c/d at P28, ~0.32 c/d at P35, ~0.32 c/d at P42), whereas mice injected beginning at P18, P21, P24 showed a decline in acuity when tested at P35 and P42. Their acuity was only slightly better than rd10-non-treated mice. Electrophysiologic function was assessed using scotopic and photopic electroretinography (ERG) to assess rod and cone function, respectively. Photopic a- and b-wave amplitudes were significantly greater in rd10 mice treated with (+)-PTZ beginning at P14 compared with non-treated mice and those in the later-onset (+)-PTZ injection groups. Retinal architecture was visualized in living mice using spectral domain-optical coherence tomography (SD-OCT) allowing measurement of the total retinal thickness, the inner retina and the outer retina (the area most affected in rd10 mice). The outer retina measured ~35 µm in rd10 mice treated with (+)-PTZ beginning at P14, which was significantly greater than mice in the later-onset (+)-PTZ injection groups (~25 µm) and non-treated rd10 mice (~25 µm). Following the visual function studies performed in the living mice, eyes were harvested at P42 for histologic analysis. While the inner retina was largely intact in all (+)-PTZ-injection groups, there was a marked reduction in the outer retina of non-treated rd10 mice (e.g. in the outer nuclear layer there were ~10 PRCs/100 µm retinal length). The rd10 mice treated with (+)-PTZ beginning at P14 had ~20 PRCs/100 µm retinal length, whereas the mice in groups beginning P18, P21 and P24 had ~16 PRCs/100 µm retinal length. In conclusion, the data indicate that delaying (+)-PTZ injection past the onset of PRC death in rd10 mice - even by a few days - can negatively impact the long-term preservation of retinal function. Our findings suggest that optimizing the administration of Sig1R ligands is critical for retinal neuroprotection.

Asunto(s)

RESUMEN

Purpose: Treatments that delay retinal cell death regardless of genetic causation are needed for inherited retinal degeneration (IRD) patients. The ketogenic diet is a high-fat, low-carbohydrate diet, used to treat epilepsy, and has beneficial effects for neurodegenerative diseases. This study aimed to determine whether the ketogenic diet could slow retinal degeneration. Methods: Early weaned, rd10 and wild-type (WT) mice were placed on either standard chow, a ketogenic diet, or a ketogenic & low-protein diet. From postnatal day (PD) 23 to PD50, weight and blood ß-hydroxybutyrate levels were recorded. Retinal thickness, retinal function, and visual performance were measured via optical coherence tomography, electroretinography (ERG), and optokinetic tracking (OKT). At PD40, serum albumin, rhodopsin protein, and phototransduction gene expression were measured. Results: Both ketogenic diets elicited a systemic induction of ketosis. However, rd10 mice on the ketogenic & low-protein diet had significant increases in photoreceptor thickness, ERG amplitudes, and OKT thresholds, whereas rd10 mice on the ketogenic diet showed no photoreceptor preservation. In both rd10 and WT mice, the ketogenic & low-protein diet was associated with abnormal weight gain and decreases in serum albumin levels, 27% and 56%, respectively. In WT mice, the ketogenic & low-protein diet was also associated with an ∼20% to 30% reduction in ERG amplitudes. Conclusions: The ketogenic & low-protein diet slowed retinal degeneration in a clinically relevant IRD model. In WT mice, the ketogenic & low-protein diet was associated with a decrease in phototransduction and serum albumin, which could serve as a protective mechanism in the rd10 model. Although ketosis was associated with protection, its role remains unclear. Translational Relevance: Neuroprotective mechanisms associated with the ketogenic & low-protein diet have potential to slow retinal degeneration.

Asunto(s)

RESUMEN

Retinitis Pigmentosa (RP) is a class of inherited disorders caused by the progressive death of photoreceptors in the retina. RP is still orphan of an effective treatment, with increasing optimism deriving from research aimed at arresting neurodegeneration or replacing light-responsive elements. All these therapeutic strategies rely on the functional integrity of the visual system downstream of photoreceptors. Whereas the inner retinal structure and optic radiation are known to be considerably preserved at least in early stages of RP, very little is known about the visual cortex. Remarkably, it remains completely unclear whether visual cortex plasticity is still present in RP. Using a well-established murine model of RP, the rd10 mouse, we report that visual cortical circuits retain high levels of plasticity, preserving their capability of input-dependent remodelling even at a late stage of retinal degeneration.

Asunto(s)

RESUMEN

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6ß proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

Asunto(s)

RESUMEN

Sigma 1 receptor (Sig1R), a putative molecular chaperone, has emerged as a novel therapeutic target for retinal degenerative disease. Earlier studies showed that activation of Sig1R via the high-affinity ligand (+)-pentazocine ((+)-PTZ) induced profound rescue of cone photoreceptor cells in the rd10 mouse model of retinitis pigmentosa; however the mechanism of rescue is unknown. Improved cone function in (+)-PTZ-treated mice was accompanied by reduced oxidative stress and normalization of levels of NRF2, a transcription factor that activates antioxidant response elements (AREs) of hundreds of cytoprotective genes. Here, we tested the hypothesis that modulation of NRF2 is central to Sig1R-mediated cone rescue. Activation of Sig1R in 661W cone cells using (+)-PTZ induced dose-dependent increases in NRF2-ARE binding activity and NRF2 gene/protein expression, whereas silencing Sig1R significantly decreased NRF2 protein levels and increased oxidative stress, although (+)-PTZ did not disrupt NRF2-KEAP1 binding. In vivo studies were conducted to investigate whether, in the absence of NRF2, activation of Sig1R rescues cones. (+)-PTZ was administered systemically for several weeks to rd10/nrf2+/+ and rd10/nrf2-/- mice. Through post-natal day 42, cone function was significant in rd10/nrf2+/+, but minimal in rd10/nrf2-/- mice as indicated by electroretinographic recordings using natural noise stimuli, optical coherence tomography and retinal histological analyses. Immunodetection of cones was limited in (+)-PTZ-treated rd10/nrf2-/-, though considerable in (+)-PTZ-treated rd10/nrf2+/+mice. The data suggest that Sig1R-mediated cone rescue requires NRF2 and provide evidence for a previously-unrecognized relationship between these proteins.

Asunto(s)

RESUMEN

Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.

RESUMEN

This review article focuses on studies of Sigma 1 Receptor (Sigma1R) and retina . It provides a brief overview of the earliest pharmacological studies performed in the late 1990s that provided evidence of the presence of Sigma1R in various ocular tissues. It then describes work from a number of labs concerning the location of Sigma1R in several retinal cell types including ganglion, Müller glia , and photoreceptors . The role of Sigma1R ligands in retinal neuroprotection is emphasized. Early studies performed in vitro clearly showed that targeting Sigma1R could attenuate stress-induced retinal cell loss. These studies were followed by in vivo experiments. Data about the usefulness of targeting Sigma1R to prevent ganglion cell loss associated with diabetic retinopathy are reviewed. Mechanisms of Sigma1R-mediated retinal neuroprotection involving Müller cells , especially in modulating oxidative stress are described along with information about the retinal phenotype of mice lacking Sigma1R (Sigma1R -/- mice). The retina develops normally in Sigma1R -/- mice, but after many months there is evidence of apoptosis in the optic nerve head, decreased ganglion cell function and eventual loss of these cells. Additional studies using the Sigma1R -/- mice provide strong evidence that in the retina, Sigma1R plays a key role in modulating cellular stress. Recent work has shown that targeting Sigma1R may extend beyond protection of ganglion cells to include photoreceptor cell degeneration as well.

Asunto(s)

RESUMEN

Retinal degenerative diseases are major causes of untreatable blindness, and novel approaches to treatment are being sought actively. Here we explored the activation of a unique protein, sigma 1 receptor (Sig1R), in the treatment of PRC loss because of its multifaceted role in cellular survival. We used Pde6ß(rd10) (rd10) mice, which harbor a mutation in the rod-specific phosphodiesterase gene Pde6ß and lose rod and cone photoreceptor cells (PRC) within the first 6 wk of life, as a model for severe retinal degeneration. Systemic administration of the high-affinity Sig1R ligand (+)-pentazocine [(+)-PTZ] to rd10 mice over several weeks led to the rescue of cone function as indicated by electroretinographic recordings using natural noise stimuli and preservation of cone cells upon spectral domain optical coherence tomography and retinal histological examination. The protective effect appears to result from the activation of Sig1R, because rd10/Sig1R(-/-) mice administered (+)-PTZ exhibited no cone preservation. (+)-PTZ treatment was associated with several beneficial cellular phenomena including attenuated reactive gliosis, reduced microglial activation, and decreased oxidative stress in mutant retinas. To our knowledge, this is the first report that activation of Sig1R attenuates inherited PRC loss. The findings may have far-reaching therapeutic implications for retinal neurodegenerative diseases.

Asunto(s)