Implications of Neural Plasticity in Retinal Prosthesis.

Retinal degenerative diseases such as retinitis pigmentosa cause a progressive loss of photoreceptors that eventually prevents the affected person from perceiving visual sensations. The absence of a visual input produces a neural rewiring cascade that propagates along the visual system. This remodeling occurs first within the retina. Then, subsequent neuroplastic changes take place at higher visual centers in the brain, produced by either the abnormal neural encoding of the visual inputs delivered by the diseased retina or as the result of an adaptation to visual deprivation. While retinal implants can activate the surviving retinal neurons by delivering electric current, the unselective activation patterns of the different neural populations that exist in the retinal layers differ substantially from those in physiologic vision. Therefore, artificially induced neural patterns are being delivered to a brain that has already undergone important neural reconnections. Whether or not the modulation of this neural rewiring can improve the performance for retinal prostheses remains a critical question whose answer may be the enabler of improved functional artificial vision and more personalized neurorehabilitation strategies.

The timing of auditory sensory deficits in Norrie disease has implications for therapeutic intervention.

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.

Electrical response of retinal ganglion cells in an N-methyl-N-nitrosourea-induced retinal degeneration porcine model.

Retinal prosthesis is regarded as the treatment for vision restoration in the blind with retinal degeneration (RD) due to the loss of photoreceptors. A strategy for retinal prosthesis is to electrically activate surviving neurons. The retina's response to electrical stimulation in a larger RD model has not been studied yet. Therefore, in this study, we investigated electrically evoked retinal responses in a previously validated N-methyl-N-nitrosourea (MNU)-induced porcine RD model. Electrically evoked responses were evaluated based on the number of retinal ganglion cell (RGC) spikes via multichannel recordings. Stimulation pulses were applied to degenerative and wild-type retinas with pulse modulation. Compared to wild-type retinas, degenerative retinas showed higher threshold values of pulse amplitude and pulse duration. The rate of increase in the number of RGC spikes relative to stimulus intensity was lower in degenerative retinas than in normal retinas. In severely degenerated retinas, few RGCs showed electrically evoked spikes. Our results suggest that the degenerative porcine retina requires a higher charge than the normal porcine retina. In the early stage of RD, it is easier to induce RGC spikes through electrical stimulation using retinal prosthesis; however, when the degeneration is severe, there may be difficulty recovering patient vision.

Assessment of retinal oxygen metabolism, visual function, thickness and degeneration markers after variable ischemia/reperfusion in rats.

After total retinal ischemia induced experimentally by ophthalmic vessel occlusion followed by reperfusion, studies have reported alterations in retinal oxygen metabolism (MO2), delivery (DO2), and extraction fraction (OEF), as well as visual dysfunction and cell loss. In the current study, under variable durations of ischemia/reperfusion, changes in these oxygen metrics, visual function, retinal thickness, and degeneration markers (gliosis and apoptosis) were assessed and related. Additionally, the prognostic value of MO2 for predicting visual function and retinal thickness outcomes was reported. Sixty-one rats were divided into 5 groups of ischemia duration (0 [sham], 60, 90, 120, or 180 min) and 2 reperfusion durations (1 h, 7 days). Phosphorescence lifetime and blood flow imaging, electroretinography, and optical coherence tomography were performed. MO2 reduction was related to visual dysfunction, retinal thinning, increased gliosis and apoptosis after 7-days reperfusion. Impairment in MO2 after 1-h reperfusion predicted visual function and retinal thickness outcomes after 7-days reperfusion. Since MO2 can be measured in humans, findings from analogous studies may find value in the clinical setting.

Functional Availability of ON-Bipolar Cells in the Degenerated Retina: Timing and Longevity of an Optogenetic Gene Therapy.

Degenerative diseases of the retina are responsible for the death of photoreceptors and subsequent loss of vision in patients. Nevertheless, the inner retinal layers remain intact over an extended period of time, enabling the restoration of light sensitivity in blind retinas via the expression of optogenetic tools in the remaining retinal cells. The chimeric Opto-mGluR6 protein represents such a tool. With exclusive ON-bipolar cell expression, it combines the light-sensitive domains of melanopsin and the intracellular domains of the metabotropic glutamate receptor 6 (mGluR6), which naturally mediates light responses in these cells. Albeit vision restoration in blind mice by Opto-mGluR6 delivery was previously shown, much is left to be explored in regard to the effects of the timing of the treatment in the degenerated retina. We performed a functional evaluation of Opto-mGluR6-treated murine blind retinas using multi-electrode arrays (MEAs) and observed long-term functional preservation in the treated retinas, as well as successful therapeutical intervention in later stages of degeneration. Moreover, the treatment decreased the inherent retinal hyperactivity of the degenerated retinas to levels undistinguishable from healthy controls. Finally, we observed for the first time micro electroretinograms (mERGs) in optogenetically treated animals, corroborating the origin of Opto-mGluR6 signalling at the level of mGluR6 of ON-bipolar cells.

Influence of Sex on Neuroretinal Degeneration: Six-Month Follow-Up in Rats With Chronic Glaucoma.

Purpose: To evaluate differences by sex in the neuroretina of rats with chronic glaucoma over 24 weeks of follow-up, and to assess by sex the influence on neurodegeneration of different methods of inducing ocular hypertension. Methods: Forty-six Long-Evans rats-18 males and 28 females-with induced chronic glaucoma were analyzed. Glaucoma was achieved via 2 models: repeatedly sclerosing the episcleral veins (9 male/14 female) or by injecting poly(lactic-co-glycolic acid) microspheres measuring 20 to 10 µm (Ms20/10) into the anterior chamber (9 male/14 female). The IOP was measured weekly by tonometer; neuroretinal function was recorded by dark/light-adapted electroretinography at baseline and weeks 12 and 24; and structure was analyzed by optical coherence tomography using the retina posterior pole, retinal nerve fiber layer and ganglion cell layer protocols at baseline and weeks 8, 12, 18, and 24. Results: Males showed statistically significant (P < 0.05) higher IOP in both chronic glaucoma models, and greater differences were found in the episcleral model at earlier stages. Males with episclerally induced glaucoma showed a statistically higher increase in retinal thickness in optical coherence tomography recordings than females and also when comparing Ms20/10 at 12 weeks. Males showed a higher percentage of retinal nerve fiber layer thickness loss in both models. Ganglion cell layer thickness loss was only detected in the Ms20/10 model. Males exhibited worse dark/light-adapted functionality in chronic glaucoma models, which worsened in the episcleral sclerosis model at 12 weeks, than females. Conclusions: Female rats with chronic glaucoma experienced lower IOP and structural loss and better neuroretinal functionality than males. Sex and the ocular hypertension-inducing method influenced neuroretinal degeneration.

Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line.

OBJECTIVES: Cone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter. METHODS: We generated OPN1LW-EGFP/NRL-/- mice by crossing NRL-/- and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL-/- mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL-/- mice were transplanted subretinally into immunodeficient Rd1 mice. RESULTS: OPN1LW-EGFP/NRL-/- retinas were enriched with OPN1LW-EGFP+ and S-opsin+ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL-/- compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL-/- were similar as the NRL-/- with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL-/- was thinner than NRL-/- controls. The OPN1LW-EGFP/NRL-/- ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL-/- controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL-/- mice showed greater S-opsin + cone survival than suspension grafts from the same strain. CONCLUSIONS: OPN1LW-EGFP/NRL-/- retinae were enriched with S-opsin+ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin+ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.

Vitamin A cycle byproducts impede dark adaptation.

Impaired dark adaptation (DA), a defect in the ability to adjust to dimly lit settings, is a universal hallmark of aging. However, the mechanisms responsible for impaired DA are poorly understood. Vitamin A byproducts, such as vitamin A dimers, are small molecules that form in the retina during the vitamin A cycle. We show that later in life, in the human eye, these byproducts reach levels commensurate with those of vitamin A. In mice, selectively inhibiting the formation of these byproducts, with the investigational drug C20D3-vitamin A, results in faster DA. In contrast, acutely increasing these ocular byproducts through exogenous delivery leads to slower DA, with otherwise preserved retinal function and morphology. Our findings reveal that vitamin A cycle byproducts alone are sufficient to cause delays in DA and suggest that they may contribute to universal age-related DA impairment. Our data further indicate that the age-related decline in DA may be tractable to pharmacological intervention by C20D3-vitamin A.

Emerging Benefits: Pathophysiological Functions and Target Drugs of the Sigma-1 Receptor in Neurodegenerative Diseases.

The sigma-1 receptor (Sig-1R) is encoded by the SIGMAR1 gene and is a nonopioid transmembrane receptor located in the mitochondrial-associated endoplasmic reticulum membrane (MAM). It helps to locate endoplasmic reticulum calcium channels, regulates calcium homeostasis, and acts as a molecular chaperone to control cell fate and participate in signal transduction. It plays an important role in protecting neurons through a variety of signaling pathways and participates in the regulation of cognition and motor behavior closely related to neurodegenerative diseases. Based on its neuroprotective effects, Sig-1R has now become a breakthrough target for alleviating Alzheimer's disease and other neurodegenerative diseases. This article reviews the most cutting-edge research on the function of Sig-1R under normal or pathologic conditions and target drugs of the sigma-1 receptor in neurodegenerative diseases.

Three "Red Lines" for Pattern Recognition-Based Differential Diagnosis Using Optical Coherence Tomography in Clinical Practice.

BACKGROUND: Optical coherence tomography (OCT) devices for imaging of the eye are broadly available. The test is noninvasive, rapid, and well-tolerated by patients. This creates a large number of OCT images and patient referrals. Interpretation of OCT findings at the interface between neurological and ophthalmologic conditions has become a key skill in the neuro-ophthalmology service. Similar to the interpretation of visual fields, recogntion of the vertical and horizontal medians are helpful. A third "red line" is added, which will be reviewed here. EVIDENCE: Levels 1a to 5 evidence. ACQUISITION: Literature research. RESULTS: There is level 1a evidence that neurodegeneration of the brain is associated with inner retinal layer atrophy. Predominantly, this is driven by retrograde (trans-synaptic) axonal degeneration from the brain to the eye. This process typically stops at the level of the inner nuclear layer (INL). Anterograde (Wallerian) axonal degeneration from the eye to the brain can trespass the INL. The geography of atrophy and swelling of individual macular retinal layers distinguishes prechiasmal from postchiasmal pathology. The emerging patterns are a front-back "red line" at the INL; a vertical "red line" through the macula for chiasmal/postchiasmal pathology; and a horizontal "red line" through the macular for pathology pointing to the optic disc. This is summarized by illustrative case vignettes. CONCLUSIONS: The interpretation of patterns of individual retinal layer atrophy (3 "red lines") needs to be combined with recognition of localized layer thickening (edema, structural) at the macula. Certain macular patterns point to pathology at the level of the optic disc. This requires revision of the optic disc OCT and will guide need for further investigations. The 3 "red lines" proposed here may be found useful in clinical practice and the related mnemonics ("half moon," "sunset," "rainbow") for teaching.

Saffron ameliorated motor symptoms, short life span and retinal degeneration in Parkinson's disease fly models.

Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In our previous paper, we found that saffron; the stigma of Crocus sativus Linné (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. In this study, we investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in the eye of A30P fly PD model. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.

The Timecourses of Functional, Morphological, and Molecular Changes Triggered by Light Exposure in Sprague-Dawley Rat Retinas.

Retinal neurodegeneration can impair visual perception at different levels, involving not only photoreceptors, which are the most metabolically active cells, but also the inner retina. Compensatory mechanisms may hide the first signs of these impairments and reduce the likelihood of receiving timely treatments. Therefore, it is essential to characterize the early critical steps in the neurodegenerative progression to design adequate therapies. This paper describes and correlates early morphological and biochemical changes in the degenerating retina with in vivo functional analysis of retinal activity and investigates the progression of neurodegenerative stages for up to 7 months. For these purposes, Sprague-Dawley rats were exposed to 1000 lux light either for different durations (12 h to 24 h) and examined seven days afterward (7d) or for a fixed duration (24 h) and monitored at various time points following the exposure (up to 210d). Flash electroretinogram (fERG) recordings were correlated with morphological and histological analyses to evaluate outer and inner retinal disruptions, gliosis, trophic factor release, and microglial activation. Twelve hours or fifteen hours of exposure to constant light led to a severe retinal dysfunction with only minor morphological changes. Therefore, early pathological signs might be hidden by compensatory mechanisms that silence retinal dysfunction, accounting for the discrepancy between photoreceptor loss and retinal functional output. The long-term analysis showed a transient functional recovery, maximum at 45 days, despite a progressive loss of photoreceptors and coincident increases in glial fibrillary acidic protein (GFAP) and basic fibroblast growth factor-2 (bFGF-2) expression. Interestingly, the progression of the disease presented different patterns in the dorsal and ventral retina. The information acquired gives us the potential to develop a specific diagnostic tool to monitor the disease's progression and treatment efficacy.

Identification of susceptibility loci for light-induced visual impairment in rats.

Bright light exposure in animals results in the selective degeneration of the outer retina, known as "retinal photic injury" (RPI). The susceptibility to RPI differs among rat strains. WKY rats display susceptibility to RPI with extensive retinal degeneration observed in the sagittal eye specimen, whereas LEW strain rats are resistant to it, showing only slight or no degeneration. In the present study, we first established an ethological screening method using the Morris water maze to discern differential susceptibility among the living rats. WKY and LEW were crossed to produce the first filial generation (F1) offspring. Maze-trained individuals were exposed to bright, white light. The screening test results demonstrated that the susceptibility to light-induced visual impairment in rats is a dominant Mendelian susceptibility trait, as F1 rats were susceptible to visual impairment like WKY rats. Therefore, F1 rats were backcrossed with recessive LEW to produce the first backcross offspring (BC1). Subsequent recurrent backcrossing while selecting for the susceptibility, indicated a segregation ratio of ca. 24% in BC1 and BC2 generations, indicating the involvement of two or more genes in the susceptibility. Further, microsatellite analysis of BC1-to-BC4 individuals using microsatellite markers mapped two susceptibility loci on chromosome segments 5q36 and 19q11-q12, named RPI susceptibility (Rpi)1 and Rpi2, respectively. This study provides an insight into mechanisms underlying differential susceptibility, which could help decipher the mechanism underlying the onset/progression of human age-related macular degeneration.

Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells.

Stem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit+/SSEA1- (C-Kit+) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR. Light stimulation of the host photoreceptors elicits distinct neuronal responses throughout the graft RPCs. The graft RPCs and their differentiated offspring cells in inner nuclear layer synchronize their activities with the host cells and exhibit presynaptic calcium flux patterns that resemble intact retinal neurons. Once graft-to-host network is established, progressive vision loss is stabilized while control eyes continually lose vision. Therefore, transplantation of organoid-derived C-Kit+ RPCs can form functional synaptic networks within ADR and it holds promising avenue for advanced RD treatment.

Longitudinal phenotypic study of late-onset retinal degeneration due to a founder variant c.562C>A p.(Pro188Thr) in the C1QTNF5 gene.

Background: Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant retinal dystrophy related to C1QTNF5 gene variants.Materials and methods: Twenty-six patients (21-81 years) with L-ORD due to c.562C>A p.(Pro188Thr) with a mean follow-up time of 8 years (range 1-37 years) underwent an extensive ophthalmic work-up.Results: Best-corrected visual acuity (BCVA) and visual fields were maintained up to 50 to 55 years (n = 8), with a gradual decline, but conservation of functional central vision between 55 to 65 years (n = 15), followed by a steep decrease in overall visual function beyond 65 years (n = 9). Classic anterior segment findings in L-ORD of abnormally long, anteriorly inserted lens zonules were absent in most patients (n = 24/26). In contrast, findings of iris transillumination and sphincter pupillae atrophy with poor dilation were novel. Patients presented with three completely different initial fundus phenotypes: adjoining pavingstone-like atrophic patches (type 1) (n = 6/20); tiny yellow-white subretinal dots (type 2) (n = 8/20); or larger yellow, thick, round sub-RPE drusenoid deposits (type 3) (n = 4/20). Two patients had a mixed phenotype. Although different in presentation phenotype, patients eventually all progressed to a common panretinal atrophy with diffuse intraretinal pigment migration beyond the age of 65. Progression pace, and thus visual prognosis, differed depending on presentation phenotype. Specifically, type 2 appears to have a more benign course.Conclusions: Phenotypic analysis showed three distinct presenting phenotypes with a considerable intrafamilial variability both in age of onset of clinical signs and in disease progression, with a fair visual potential (>20/40) until the seventh decade.Abbreviations: L-ORD: Late-onset retinal degeneration; C1QTNF5: complement 1Q tumor necrosis factor 5; OCT: Ocular coherence tomography; BCVA: Best-corrected visual acuity; RPE: Retinal pigment epithelium; ffERG: Full-field electroretinography; IRD: Inherited retinal dystrophy; CNV: Choroidal neovascularization; LAZ: Long anteriorly inserted zonules; AMPK: AMP-activated protein kinase; IOP: Intraocular pressure; cSLO: confocal scanning laser ophthalmoscopy; BAF: Blue light autofluorescence; NIR-AF: Near-infrared autofluorescence; NIR-R: Near-infrared reflectance; RF: Red-free; SD-OCT: Spectral domain ocular coherence tomography; HRR: Hardy-Rand-Rittler pseudo-isochromatic plates; AS: anterior segment; UBM: ultrasound biomicroscopy; PCR: Polymerase chain reaction; SNP: Single nucleotide polymorphism; VEGF: Vascular endothelial growth factor; IZ: Interdigitation zone; EZ: Ellipsoid zone; ELM: External limiting membrane; LP: Light perception; AMD: Age-related macular degeneration; SFD: Sorsby fundus dystrophy.

Age-dependent favorable visual recovery despite significant retinal atrophy in pediatric MOGAD: how much retina do you really need to see well?

BACKGROUND: To investigate age-related severity, patterns of retinal structural damage, and functional visual recovery in pediatric and adult cohorts of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) optic neuritis (ON). METHODS: All MOGAD patients from the 5 participating centers were included. Patients with initial manifestation <18 years were included in the pediatric (MOGADped) cohort and patients with ≥18 years in the adult (MOGADadult) cohort. For patients with MOGAD ON, examinations at least ≥6 months after ON onset were included in the analyses. Using spectral domain optical coherence tomography (SD-OCT), we acquired peripapillary retinal nerve fiber layer thickness (pRNFL) and volumes of combined ganglion cell and inner plexiform layer (GCIPL). High- and 2.5% low-contrast visual acuity (HCVA, LCVA) and visual-evoked potentials (VEP) were obtained. RESULTS: Twenty MOGADped (10.3±3.7 years, 30 MOGAD ON eyes) and 39 MOGADadult (34.9±11.6 years, 42 MOGAD ON eyes) patients were included. The average number of ON episodes per ON eye was similar in both groups (1.8±1.3 and 2.0±1.7). In both pediatric and adult MOGAD, ON led to pronounced neuroaxonal retinal atrophy (pRNFL: 63.1±18.7 and 64.3±22.9 µm; GCIPL: 0.42±0.09 and 0.44±0.13 mm3, respectively) and moderate delay of the VEP latencies (117.9±10.7 and 118.0±14.5 ms). In contrast, visual acuity was substantially better in children (HCVA: 51.4±9.3 vs. 35.0±20.6 raw letters, p=0.001; LCVA: 22.8±14.6 vs. 13.5±16.4, p=0.028). Complete visual recovery (HCVA-logMAR 0.0) occurred in 73.3% of MOGADped and 31% MOGADadults ON eyes, while 3.3% and 31% demonstrated moderate to severe (logMAR > 0.5) visual impairment. Independent of retinal atrophy, age at ON onset significantly correlated with visual outcome. CONCLUSION: Pediatric MOGAD ON showed better visual recovery than adult MOGAD ON despite profound and almost identical neuroaxonal retinal atrophy. Age-related cortical neuroplasticity may account for the substantial discrepancy between structural changes and functional outcomes.

Development and characterization of a chronic photoreceptor degeneration model in adult zebrafish that does not trigger a regenerative response.

Zebrafish (Danio rerio) have become a highly-utilized model system in the field of regenerative biology because of their endogenous ability to regenerate many tissues and organs, including the retina. The vast majority of previous research on retinal regeneration in adult zebrafish utilizes acute methodologies for retinal damage. Acute retinal cell death triggers a reactive gliosis response of Müller glia (MG), the resident macroglia of the retina. In addition, each activated MG undergoes asymmetric cell division to produce a neuronal progenitor, which continues to divide and ultimately gives rise to new retinal neurons. Studies using these approaches have uncovered many crucial mechanisms by which MG respond to acute damage. However, they may not adequately mimic the chronic neuronal degeneration observed in many human retinal degenerative diseases. The current study aimed to develop a new long-term, chronic photoreceptor damage and degeneration model in adult zebrafish. Comparing the subsequent cellular responses to that of the commonly-used acute high-intensity model, we found that low, continuous light exposure damaged the outer segments of both rod and cone photoreceptors, but did not result in significant apoptotic cell death, MG gliosis, or MG cell-cycle re-entry. Instead, chronic light nearly completely truncated photoreceptor outer segments and resulted in a recruitment of microglia to the area. Together, these studies present a chronic photoreceptor model that can be performed in a relatively short time frame (21 days), that may lend insight into the cellular events underlying non-regenerative photoreceptor degeneration observed in other model systems.

Synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model.

Purpose: To explore synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model. Methods: Sprague-Dawley rats were euthanized 1 h, 1 day, 3 days, 7 days, and 14 days after being exposed to intense blue light for 24 h. Hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were used to evaluate changes in the outer nuclear layer (ONL). Transmission electron microscopy (TEM) was applied to observe the ultrastructural changes in the synapses between the photoreceptors and second-order neurons. Western blotting was conducted to evaluate specific proteins, including postsynaptic density-95 (PSD-95), metabotropic glutamate receptor 6 (mGluR6), synapsin I, and synaptophysin. Immunofluorescence of CD11b and PKC-α or mGluR6 was used to explore the spatial relationships between microglial processes and synaptic elements. Immunoelectron microscopy of PSD-95 was performed to further confirm its engulfment of synaptic materials. Results: H&E and TUNEL staining showed that the thickness of the ONL decreased markedly, and the number of apoptotic photoreceptors peaked at day 1. TEM revealed darkened photoreceptor terminals and that ribbons of them were floating in the cytoplasm, coinciding with the downregulation of PSD-95 and mGluR6. Downstream synaptic protein synapsin I and synaptophysin exhibited upregulation in the inner plexiform layer. Activated microglia migrated to the outer retina, and their processes were found in close proximity to synapses in the outer plexiform layer under light and electron microscopy levels. Double immunostaining of CD11b and mGluR6 showed colocalization. PSD-95-immunoreactive electron-dense materials were observed inside the microglia suggesting engulfment of synaptic components. Conclusions: The study showed that there are early synaptic impairment and late compensatory changes in downstream synapses in this photic injury model. Activated microglia touched and directly engulfed synaptic materials. Microglia may play a role or a partial role in synaptic changes.

Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5-/- or CLN6-/- animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5-/- animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6-/- animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5-/- animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5-/- sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.

Noncoding mutation in RPGRIP1 contributes to inherited retinal degenerations.

Purpose: Despite the extensive use of next-generation sequencing (NGS) technology to identify disease-causing genomic variations, a major gap in our understanding of Mendelian diseases is the unidentified molecular lesion in a significant portion of patients. For inherited retinal degenerations (IRDs), although currently close to 300 disease-associated genes have been identified, the mutations in approximately one-third of patients remain unknown. With mounting evidence that noncoding mutations might contribute significantly to disease burden, we aimed to systematically investigate the contributions of noncoding regions in the genome to IRDs. Methods: In this study, we focused on RPGRIP1, which has been linked to various IRD phenotypes, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and macular dystrophy (MD). As several noncoding mutant alleles have been reported in RPGRIP1, and we observed that the mutation carrier frequency of RPGRIP1 is higher in patient cohorts with unsolved IRDs, we hypothesized that mutations in the noncoding regions of RPGRIP1 might be a significant contributor to pathogenicity. To test this hypothesis, we performed whole-genome sequencing (WGS) for 25 patients with unassigned IRD who carry a single mutation in RPGRIP1. Results: Three noncoding variants in RPGRIP1, including a 2,890 bp deletion and two deep-intronic variants (c.2710+233G>A and c.1468-263G>C), were identified as putative second hits of RPGRIP1 in three patients with LCA. The mutant alleles were validated with direct sequencing or in vitro assays. Conclusions: The results highlight the significance of the contribution of noncoding pathogenic variants to unsolved IRD cases.