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PURPOSE: Targeted drug delivery to the optic nerve head may be useful in the preclinical study and later clinical management of optic neuropathies, however, there are no FDA-approved drug delivery systems to achieve this. The purpose of this work was to develop an optic nerve head drug delivery technique. METHODS: Different strategies to approach the optic nerve head were investigated, including standard intravitreal and retroorbital injections. A novel SupraChoroidal-to-Optic-NervE (SCONE) delivery was optimized by creating a sclerotomy and introducing a catheter into the suprachoroidal space. Under direct visualization, the catheter was guided to the optic nerve head. India ink was injected. The suprachoroidal approach was performed in New Zealand White rabbit eyes in vivo (25 animals total). Parameters, including microneedle size and design, catheter design, and catheter tip angle, were optimized ex vivo and in vivo. RESULTS: Out of the candidate optic nerve head approaches, intravitreal, retroorbital, and suprachoroidal approaches were able to localize India ink to within 2 mm of the optic nerve. The suprachoroidal approach was further investigated, and after optimization, was able to deposit India ink directly within the optic nerve head in up to 80% of attempts. In eyes with successful SCONE delivery, latency and amplitude of visual evoked potentials was not different than the naïve untreated eye. CONCLUSIONS: SCONE delivery can be used for targeted drug delivery to the optic nerve head of rabbits without measurable toxicity measured anatomically or functionally. Successful development of this system may yield novel opportunities to study optic nerve head-specific drug delivery in animal models, and paradigm-shifting management strategies for treating optic neuropathies. TRANSLATIONAL RELEVANCE: Here we demonstrate data on a new method for targeted delivery to the optic nerve head, addressing a significant unmet need in therapeutics for optic neuropathies.
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Sistemas de Liberación de Medicamentos , Animales , Conejos , Coroides , Nervio Óptico/efectos de los fármacos , Potenciales Evocados Visuales/efectos de los fármacos , Disco Óptico , Inyecciones Intravítreas , Agujas , CarbonoRESUMEN
Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.
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Mitocondrias , Oxidorreductasas , Proteínas de Plantas , Epitelio Pigmentado de la Retina , Animales , Mitocondrias/metabolismo , Ratones , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Ciona intestinalis/metabolismo , Ubiquinona/metabolismo , Ubiquinona/análogos & derivados , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologíaRESUMEN
In patients with atrophic age-related macular degeneration, subretinal photovoltaic implant (PRIMA) provided visual acuity up to 20/440, matching its 100µm pixels size. Next-generation implants with smaller pixels should significantly improve the acuity. This study in rats evaluates removal of a subretinal implant, replacement with a newer device, and the resulting grating acuity in-vivo. Six weeks after the initial implantation with planar and 3-dimensional devices, the retina was re-detached, and the devices were successfully removed. Histology demonstrated a preserved inner nuclear layer. Re-implantation of new devices into the same location demonstrated retinal re-attachment to a new implant. New devices with 22µm pixels increased the grating acuity from the 100µm capability of PRIMA implants to 28µm, reaching the limit of natural resolution in rats. Reimplanted devices exhibited the same stimulation threshold as for the first implantation of the same implants in a control group. This study demonstrates the feasibility of safely upgrading the subretinal photovoltaic implants to improve prosthetic visual acuity.
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Traumatic optic neuropathy (TON) is a devastating condition that can occur after blunt or penetrating trauma to the head, leading to visual impairment or blindness. Despite these debilitating effects, no clinically available therapeutic targets neuroprotection or promotes axon regeneration in this or any optic neuropathy. Limited data in large-animal models are a major obstacle to advancing treatments toward clinical therapeutics. To address this issue, we refined a surgical model of TON in Yucatan minipigs. First, we validated the model by demonstrating visual impairment by flash visual-evoked potential and retinal ganglion cell degeneration and death. Next, we developed and optimized a delivery method and nontoxic dosing of intravitreal brain-derived neurotrophic factor (BDNF) and cAMP. Finally, we showed that intravitreal injection of BDNF and cAMP rescued visual function and protected against retinal ganglion cell death and optic nerve axon degeneration. Together these data in a preclinical large-animal model advance our understanding of and ability to model TON and further identify and develop candidate clinical therapeutics.
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Factor Neurotrófico Derivado del Encéfalo , Traumatismos del Nervio Óptico , Animales , Porcinos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Traumatismos del Nervio Óptico/tratamiento farmacológico , Axones/metabolismo , Neuroprotección , Regeneración Nerviosa , Porcinos Enanos/metabolismo , Trastornos de la VisiónRESUMEN
Purpose: Verteporfin is a benzoporphyrin derivative which is Food and Drug Administration-approved for treatment of choroidal neovascularization in conjunction with photodynamic therapy. It has been shown to prevent fibrosis and scar formation in several organs and represents a promising novel antifibrotic agent for glaucoma surgery. The goal of this study is to determine the effect of verteporfin on wound healing after glaucoma filtration surgery. Design: Preclinical study using a rabbit model of glaucoma filtration surgery. Subjects: Eight New Zealand white rabbits underwent glaucoma filtration surgery in both eyes. Methods: Eyes were randomized into 4 study groups to receive a postoperative subconjunctival injection of 1 mg/mL verteporfin (n = 4), 0.4 mg/mL mitomycin C (MMC; n = 4), 0.4 mg/mL MMC + 1 mg/mL verteporfin (n = 4), or balanced salt solution (BSS) control (n = 4). Bleb survival, vascularity, and morphology were graded using a standard scale over a 30-day period, and intraocular pressure (IOP) was monitored. At 30 days postoperative or surgical failure, histology was performed to evaluate for inflammation, local toxicity, and scarring. Main Outcome Measures: The primary outcome measure was bleb survival. Secondary outcome measures were IOP, bleb morphology, and bleb histology. Results: Compared to BSS control blebs, verteporfin-treated blebs demonstrated a trend toward increased surgical survival (mean 9.8 vs. 7.3 days, log rank P = 0.08). Mitomycin C-treated blebs survived significantly longer than verteporfin-treated blebs (log rank P = 0.009), with all but 1 MMC-treated bleb still surviving at postoperative day 30. There were no significant differences in survival between blebs treated with combination verteporfin + MMC and MMC alone. Mitomycin C-treated blebs were less vascular than verteporfin-treated blebs (mean vascularity score 0.3 ± 0.5 for MMC vs. 1.0 ± 0.0 for verteporfin, P < 0.01). Bleb histology did not reveal any significant toxicity in verteporfin-treated eyes. There were no significant differences in inflammation or scarring across groups. Conclusions: Although verteporfin remained inferior to MMC with regard to surgical survival, there was a trend toward increased survival compared with BSS control and it had an excellent safety profile. Further studies with variations in verteporfin dosage and/or application frequency are needed to assess whether this may be a useful adjunct to glaucoma surgery. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.
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Astrocitos , Neuroprotección , Adenilil Ciclasas/metabolismo , Astrocitos/citología , Astrocitos/enzimología , Astrocitos/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Supervivencia Celular , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Microglía/metabolismo , Microglía/patología , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Traumatismos del Nervio Óptico/terapia , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Glaucoma/patología , Glaucoma/terapiaRESUMEN
PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.
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Glaucoma , Canales Iónicos , Animales , Ratones , Glaucoma/genética , Hibridación Fluorescente in Situ , Canales Iónicos/metabolismo , Mecanotransducción Celular , Ratones Transgénicos , ARN Mensajero/genéticaRESUMEN
Optic neuropathy is a group of optic nerve (ON) diseases with progressive degeneration of ON and retinal ganglion cells (RGCs). The lack of neuroprotective treatments is a central challenge for this leading cause of irreversible blindness. SARM1 (sterile α and TIR motif-containing protein 1) has intrinsic nicotinamide adenine dinucleotide (NAD+) hydrolase activity that causes axon degeneration by degrading axonal NAD+ significantly after activation by axon injury. SARM1 deletion is neuroprotective in many, but not all, neurodegenerative disease models. Here, we compare two therapy strategies for SARM1 inhibition, antisense oligonucleotide (ASO) and CRISPR, with germline SARM1 deletion in the neuroprotection of three optic neuropathy mouse models. This study reveals that, similar to germline SARM1 knockout in every cell, local retinal SARM1 ASO delivery and adeno-associated virus (AAV)-mediated RGC-specific CRISPR knockdown of SARM1 provide comparable neuroprotection to both RGC somata and axons in the silicone oil-induced ocular hypertension (SOHU) glaucoma model but only protect RGC axons, not somata, after traumatic ON injury. Surprisingly, neither of these two therapy strategies of SARM1 inhibition nor SARM1 germline knockout (KO) benefits RGC or ON survival in the experimental autoimmune encephalomyelitis (EAE)/optic neuritis model. Our studies therefore suggest that SARM1 inhibition by local ASO delivery or AAV-mediated CRISPR is a promising neuroprotective gene therapy strategy for traumatic and glaucomatous optic neuropathies but not for demyelinating optic neuritis.
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Previously, we developed a simple procedure of intracameral injection of silicone oil (SO) into mouse eyes and established the mouse SOHU (SO-induced ocular hypertension under-detected) glaucoma model with reversible intraocular pressure (IOP) elevation and significant glaucomatous neurodegeneration. Because the anatomy of the non-human primate (NHP) visual system closely resembles that of humans, it is the most likely to predict human responses to diseases and therapies. Here we tried to replicate the mouse SOHU glaucoma model in rhesus macaque monkeys. All six animals that we tested showed significant retinal ganglion cell (RGC) death, optic nerve (ON) degeneration, and visual functional deficits at both 3 and 6 months. In contrast to the mouse SOHU model, however, IOP changed dynamically in these animals, probably due to individual differences in ciliary body tolerance capability. Further optimization of this model is needed to achieve consistent IOP elevation without permanent damage of the ciliary body. The current form of the NHP SOHU model recapitulates the severe degeneration of acute human glaucoma, and is therefore suitable for assessing experimental therapies for neuroprotection and regeneration, and therefore for translating relevant findings into novel and effective treatments for patients with glaucoma and other neurodegenerations.
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Glaucoma , Hipertensión Ocular , Humanos , Ratones , Animales , Macaca mulatta , Aceites de Silicona , Glaucoma/metabolismo , Hipertensión Ocular/inducido químicamente , Hipertensión Ocular/metabolismo , Presión Intraocular , Modelos Animales de EnfermedadRESUMEN
Nonarteritic anterior ischemic optic neuropathy (NAION) is a common acute optic neuropathy and cause of irreversible vision loss in those older than 50 years of age. There is currently no effective treatment for NAION and the biological mechanisms leading to neuronal loss are not fully understood. Promising novel targets include glial cells activation and intercellular communication mediated by molecules such as gap junction protein Connexin 43 (Cx43), which modulate neuronal fate in central nervous system disorders. In this study, we investigated retinal glial changes and neuronal loss following a novel NAION animal model using a 577â¯nm yellow laser. We induced unilateral photochemical thrombosis using rose bengal at the optic nerve head vasculature in adult C57BL/6 mice using a 577â¯nm laser and performed morphometric analysis of the retinal structure using serial in vivo optical coherence tomography (OCT) and histology for glial and neuronal markers. One day after experimental NAION, in acute phase, OCT imaging revealed peripapillary thickening of the retinal ganglion cell complex (GCC, baseline: 79.5⯱â¯1.0⯵m, nâ¯=â¯8; NAION: 93.0⯱â¯2.5⯵m, nâ¯=â¯8, Pâ¯<â¯0.01) and total retina (baseline: 202.9⯱â¯2.4⯵m, nâ¯=â¯8; NAION: 228.1⯱â¯6.8⯵m, nâ¯=â¯8, Pâ¯<â¯0.01). Twenty-one days after ischemia, at a chronic phase, there was significant GCC thinning (baseline 78.3⯱â¯2.1⯵m, nâ¯=â¯6; NAION: 72.2⯱â¯1.9⯵m, nâ¯=â¯5, Pâ¯<â¯0.05), mimicking human disease. Examination of molecular changes in the retina one day after ischemia revealed that NAION induced a significant increase in retinal VEGF levels (control: 2319⯱â¯195, nâ¯=â¯5; NAION: 4549⯱â¯683 gray mean value, nâ¯=â¯5, Pâ¯<â¯0.05), which highly correlated with retinal thickness (râ¯=â¯0.89, Pâ¯<â¯0.05). NAION also led to significant increase in mRNA level for Cx43 (Gj1a) at day 1 (control: 1.291⯱â¯0.38; NAION: 3.360⯱â¯0.58 puncta/mm2, nâ¯=â¯5, Pâ¯<â¯0.05), but not of glial fibrillary acidic protein (Gfap) at the same time (control: 2,800⯱â¯0.59; NAION: 4,690⯱â¯0.90 puncta/mm2 nâ¯=â¯5, Pâ¯=â¯0.19). Retinal ganglion cell loss at day 21 was confirmed by a 30% decrease in Brn3a+ cells (control: 2,844⯱â¯235; NAION: 2,001⯱â¯264â¯cells/mm2, n = 4, P < 0.05). We described a novel protocol of NAION induction by photochemical thrombosis using a 577 nm laser, leading to retinal edema and VEGF increase at day 1 and RGCs loss at day 21 after injury, consistent with the pathophysiology of human NAION. Early changes in glial cells intercommunication revealed by increased Cx43+ gap junctions are consistent with a retinal glial role in mediating cell-to-cell signaling after an ischemic insult. Our study demonstrates an early glial response in a novel NAION animal model and reveals glial intercommunication molecules such as Cx43 as a promising therapeutic target in acute NAION.
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Neuropatía Óptica Isquémica , Adulto , Ratones , Humanos , Animales , Neuropatía Óptica Isquémica/diagnóstico , Factor A de Crecimiento Endotelial Vascular , Conexina 43/genética , Regulación hacia Arriba , Ratones Endogámicos C57BL , Retina/patología , Tomografía de Coherencia Óptica/métodos , Rayos Láser , Modelos Animales de EnfermedadRESUMEN
Optic neuritis, inflammation, and demyelination of the optic nerve (ON), is one of the most common clinical manifestations of multiple sclerosis; affected patients suffer persistent visual symptoms due to ON degeneration and secondary retinal ganglion cell (RGC) death. The mouse experimental autoimmune encephalomyelitis (EAE) model replicates optic neuritis and significant RGC soma and axon loss. Nicotinamide mononucleotide adenylyltransferases (NMNATs) are NAD+-synthetic enzymes that have been shown to be essential for axon integrity, activation of which significantly delays axonal Wallerian degeneration. NMNAT2, which is enriched in axons, has been proposed as a promising therapeutic target for axon injury-induced neurodegeneration. We therefore investigated whether activation of NMNAT2 can be used as a gene therapy strategy for neuroprotection in EAE/optic neuritis. To avoid the confounding effects in inflammatory cells, which play important roles in EAE initiation and progression, we used an RGC-specific promoter to drive the expression of the long half-life NMNAT2 mutant in mouse RGCs in vivo. However, optical coherence tomography in vivo retina imaging did not reveal significant protection of the ganglion cell complex, and visual function assays, pattern electroretinography, and optokinetic response also showed no improvement in mice with NMNAT2 overexpression. Postmortem histological analysis of retina wholemounts and semithin sections of ON confirmed the in vivo results: NMNAT2 activation in RGCs does not provide significant neuroprotection of RGCs in EAE/optic neuritis. Our studies suggest that a different degenerative mechanism than Wallerian degeneration is involved in autoimmune inflammatory axonopathy and that NMNAT2 may not be a major contributor to this mechanism.
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Recently, we established silicone oil-induced ocular hypertension (SOHU) mouse model with significant glaucomatous neurodegeneration. Here we characterize two additional variations of this model that simulate two distinct glaucoma types. The first is a chronic model produced by high frequency (HF) pupillary dilation after SO-induced pupillary block, which shows sustained moderate IOP elevation and corresponding slow, mild glaucomatous neurodegeneration. We also demonstrate that although SO removal quickly returns IOP to normal, the glaucomatous neurodegeneration continues to advance to a similar degree as in the HF group without SO removal. The second, an acute model created by no pupillary dilation (ND), shows a greatly elevated IOP and severe inner retina degeneration at an early time point. Therefore, by a straightforward dilation scheme, we extend our original SOHU model to recapitulate phenotypes of two major glaucoma forms, which will be invaluable for selecting neuroprotectants and elucidating their molecular mechanisms.
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Modelos Animales de Enfermedad , Glaucoma/patología , Hipertensión Ocular/fisiopatología , Degeneración Retiniana/patología , Células Ganglionares de la Retina/patología , Aceites de Silicona/toxicidad , Enfermedad Aguda , Animales , Femenino , Glaucoma/inducido químicamente , Presión Intraocular , Masculino , Ratones , Ratones Endogámicos C57BL , Hipertensión Ocular/inducido químicamente , Degeneración Retiniana/inducido químicamente , Células Ganglionares de la Retina/efectos de los fármacos , Aceites de Silicona/administración & dosificaciónRESUMEN
Central nervous system and visual dysfunction is an unfortunate consequence of systemic hypoxia in the setting of cardiopulmonary disease, including infection with SARS-CoV-2, high-altitude cerebral edema and retinopathy and other conditions. Hypoxia-induced inflammatory signaling may lead to retinal inflammation, gliosis and visual disturbances. We investigated the consequences of systemic hypoxia using serial retinal optical coherence tomography and by assessing the earliest changes within 24h after hypoxia by measuring a proteomics panel of 39 cytokines, chemokines and growth factors in the plasma and retina, as well as using retinal histology. We induced severe systemic hypoxia in adult C57BL/6 mice using a hypoxia chamber (10% O2) for 1 week and rapidly assessed measurements within 1h compared with 18h after hypoxia. Optical coherence tomography revealed retinal tissue edema at 18h after hypoxia. Hierarchical clustering of plasma and retinal immune molecules revealed obvious segregation of the 1h posthypoxia group away from that of controls. One hour after hypoxia, there were 10 significantly increased molecules in plasma and 4 in retina. Interleukin-1ß and vascular endothelial growth factor were increased in both tissues. Concomitantly, there was significantly increased aquaporin-4, decreased Kir4.1, and increased gliosis in retinal histology. In summary, the immediate posthypoxic period is characterized by molecular changes consistent with systemic and retinal inflammation and retinal glial changes important in water transport, leading to tissue edema. This posthypoxic inflammation rapidly improves within 24h, consistent with the typically mild and transient visual disturbance in hypoxia, such as in high-altitude retinopathy. Given hypoxia increases risk of vision loss, more studies in at-risk patients, such as plasma immune profiling and in vivo retinal imaging, are needed in order to identify novel diagnostic or prognostic biomarkers of visual impairment in systemic hypoxia.
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Hipoxia/complicaciones , Inflamación/etiología , Retina/patología , Animales , Sistema Nervioso Central/patología , Citocinas/análisis , Citocinas/sangre , Femenino , Hipoxia/sangre , Hipoxia/patología , Inflamación/sangre , Inflamación/patología , Péptidos y Proteínas de Señalización Intercelular/análisis , Péptidos y Proteínas de Señalización Intercelular/sangre , Masculino , Ratones Endogámicos C57BLRESUMEN
Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma, and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno-associated virus (AAV)-mediated gene transfer to perform a low-throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can knock down pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.SIGNIFICANCE STATEMENT Here, we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve (ON) by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.
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Regulación de la Expresión Génica/genética , Edición de ARN/genética , Células Ganglionares de la Retina/metabolismo , gamma-Sinucleína/genética , Animales , Sistemas CRISPR-Cas , Dependovirus/genética , Femenino , Eliminación de Gen , Terapia Genética , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Ratones , Ratones Endogámicos C57BL , Nervio Óptico/patología , Enfermedades del Nervio Óptico/genética , Enfermedades del Nervio Óptico/terapia , Células Ganglionares de la Retina/patología , Transgenes/genéticaRESUMEN
Vision loss is a devastating consequence of systemic hypoxia, but the cellular mechanisms are unclear. We investigated the impact of acute hypoxia in the retina and optic nerve. We induced systemic hypoxia (10% O2) in 6-8w mice for 48 h and performed in vivo imaging using optical coherence tomography (OCT) at baseline and after 48 h to analyze structural changes in the retina and optic nerve. We analyzed glial cellular and molecular changes by histology and immunofluorescence and the impact of pretreatment with 4-phenylbutyric acid (4-PBA) in oligodendroglia survival. After 48 h hypoxia, we found no change in ganglion cell complex thickness and no loss of retinal ganglion cells. Despite this, there was significantly increased expression of CCAAT-enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum stress, in the retina and optic nerve. In addition, hypoxia induced obvious increase of GFAP expression in the anterior optic nerve, where it co-localized with CHOP, and significant loss of Olig2+ oligodendrocytes. Pretreatment with 4-PBA, which has been shown to reduce endoplasmic reticulum stress, rescued total Olig2+ oligodendrocytes and increased the pool of mature (CC-1+) but not of immature (PDGFRa+) oligodendrocytes. Consistent with a selective vulnerability of the retina and optic nerve in hypoxia, the most striking changes in the 48 h murine model of hypoxia were in glial cells in the optic nerve, including increased CHOP expression in the astrocytes and loss of oligodendrocytes. Our data support a model where glial dysfunction is among the earliest events in systemic hypoxia - suggesting that glia may be a novel target in treatment of hypoxia.
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Hipoxia/complicaciones , Neuroglía/patología , Enfermedades del Nervio Óptico/diagnóstico , Nervio Óptico/patología , Animales , Supervivencia Celular , Modelos Animales de Enfermedad , Femenino , Hipoxia/diagnóstico , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Enfermedades del Nervio Óptico/etiología , Células Ganglionares de la Retina/patología , Tomografía de Coherencia Óptica/métodosRESUMEN
PURPOSE: To evaluate and compare the effect of lacrimal nerve stimulation (LNS) and anterior ethmoid nerve stimulation (AENS) on aqueous tear secretion, and tissue condition following chronic implantation. METHODS: A neurostimulator was implanted in rabbits adjacent to the (1) lacrimal nerve, and (2) anterior ethmoid nerve. Tear volume was measured with Schirmer test strips after stimulation (2.3-2.8â¯mA pulses at 30â¯Hz for 3-5â¯min), and scores were compared to sham stimulation. Lacrimal gland and nasal septal tissue were evaluated histologically after chronic stimulation (2 weeks-7 months). RESULTS: LNS increases tear volume by 32% above sham (pâ¯<â¯0.05, nâ¯=â¯5), compared with 133% for AENS (pâ¯≤â¯0.01, nâ¯=â¯6). AENS also significantly increases tear secretion in the fellow, non-stimulated eye (pâ¯≤â¯0.01, nâ¯=â¯6), as expected from the tearing reflex pathway. Histologically, chronic LNS is well tolerated by surrounding tissues while chronic AENS results in nasal mucosal fibrosis and implant extrusion within 3 weeks. CONCLUSIONS: AENS is significantly more effective than LNS at enhancing aqueous tear secretion, including the fellow eye. The lacrimal implant is well tolerated, while the nasal implant requires further design optimization to improve tolerability.
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Síndromes de Ojo Seco/terapia , Estimulación Eléctrica/métodos , Aparato Lagrimal/inervación , Lágrimas/metabolismo , Animales , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/fisiopatología , Aparato Lagrimal/metabolismo , Masculino , Nervio Oftálmico , ConejosRESUMEN
PURPOSE: We investigated the effects of various retinal laser therapies on preservation of the photoreceptors in an animal model of Mer tyrosine kinase receptor (MERTK)-related retinitis pigmentosa (RP). These modalities included photocoagulation with various pattern densities, selective RPE therapy (SRT), and nondamaging retinal therapy (NRT). METHODS: Laser treatments were performed on right eyes of RCS rats, using one of three laser modalities. For photocoagulation, six pattern densities (spot spacings of 0.5, 1, 1.5, 3, 4, and 5 spot diameters) were delivered in 19-day-old animals, prior to the onset of photoreceptor degeneration, to determine the optimal treatment density for the best preservation of photoreceptors. The left eye was used as control. Rats were monitored for 6 months after treatment using electroretinography, optical coherence tomography, and histology. RESULTS: Photocoagulation resulted in long-term preservation of photoreceptors, manifested morphologically and functionally, with the extent of the benefit dependent on the laser pattern density. Eyes treated with a 1.5 spot size spacing showed the best morphologic and functional preservation during the 6-month follow-up. SRT-treated eyes exhibited short-term morphologic preservation, but no functional benefit. NRT-treated eyes did not show any observable preservation benefit from the treatment. CONCLUSIONS: In a rodent model of MERTK-related RP, pattern photocoagulation of about 15% of the photoreceptors (1.5 spot diameter spacing) provides long-term preservation of photoreceptors in the treatment area. TRANSLATIONAL RELEVANCE: Availability of retinal lasers in ophthalmic practice enables rapid translation of our study to clinical testing and may help preserve the sight in patients with photoreceptor degeneration.
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High-resolution visual prostheses require small, densely packed pixels, but limited penetration depth of the electric field formed by a planar electrode array constrains such miniaturization. We present a novel honeycomb configuration of an electrode array with vertically separated active and return electrodes designed to leverage migration of retinal cells into voids in the subretinal space. Insulating walls surrounding each pixel decouple the field penetration depth from the pixel width by aligning the electric field vertically, enabling a decrease of the pixel size down to cellular dimensions. We demonstrate that inner retinal cells migrate into the 25 µm deep honeycomb wells as narrow as 18 µm, resulting in more than half of these cells residing within the electrode cavities. Immune response to honeycombs is comparable to that with planar arrays. Modeled stimulation threshold current density with honeycombs does not increase substantially with reduced pixel size, unlike quadratic increase with planar arrays. This 3-D electrode configuration may enable functional restoration of central vision with acuity better than 20/100 for millions of patients suffering from age-related macular degeneration.
Asunto(s)
Neuronas/fisiología , Diseño de Prótesis , Retina/cirugía , Degeneración Retiniana/cirugía , Prótesis Visuales , Animales , Modelos Animales de Enfermedad , Electrodos Implantados , Implantación de Prótesis , Ratas , Retina/fisiopatología , Degeneración Retiniana/fisiopatologíaRESUMEN
PURPOSE: To demonstrate survival and integration of mature photoreceptors transplanted with the retinal pigment epithelium (RPE). METHODS: Full-thickness retina with attached RPE was harvested from healthy adult rats. Grafts were implanted into two rat models of retinal degeneration, Royal College of Surgeons (RCS) and S334ter-3. Survival of the host and transplanted retina was monitored using optical coherence tomography (OCT) for up to 6 months. The retinal structure and synaptogenesis between the host and transplant was assessed by histology and immunohistochemistry. RESULTS: OCT and histology demonstrated a well-preserved photoreceptor layer with inner and outer segments, while the inner retinal layers of the transplant largely disappeared. Grafts, including RPE, survived better than without and the transplanted RPE appeared as a monolayer integrated with the native one. Synaptogenesis was observed through sprouting of new dendrites from the host bipolar cells and synaptic connections forming with cells of the transplant. However, in many samples, a glial fibrillary acidic protein-positive membrane separated the host retina and the graft. CONCLUSIONS: Presence of RPE in the graft improved the survival of transplanted photoreceptors. Functional integration between the transplant and the host retina is likely to be further enhanced if formation of a glial seal could be prevented. Transplantation of the mature photoreceptors with RPE may be a practical approach to restoration of sight in retinal degeneration. TRANSLATIONAL RELEVANCE: This approach to restoration of sight in patients with photoreceptor degeneration can be rapidly advanced to clinical testing. In patients with central scotoma, autologous transplantation of the peripheral retina can be an option.
RESUMEN
Purpose: Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation. Methods: Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining. Results: Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study. Conclusions: Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.