RESUMEN
Pathogenic variants in HCN1 are associated with a range of epilepsy syndromes including a developmental and epileptic encephalopathy. The recurrent de novo HCN1 pathogenic variant (M305L) results in a cation leak, allowing the flux of excitatory ions at potentials where the wild-type channels are closed. The Hcn1M294L mouse recapitulates patient seizure and behavioral phenotypes. As HCN1 channels are highly expressed in rod and cone photoreceptor inner segments, where they shape the light response, mutated channels are likely to impact visual function. Electroretinogram (ERG) recordings from male and female mice Hcn1M294L mice revealed a significant decrease in the photoreceptor sensitivity to light, as well as attenuated bipolar cell (P2) and retinal ganglion cell responses. Hcn1M294L mice also showed attenuated ERG responses to flickering lights. ERG abnormalities are consistent with the response recorded from a single female human subject. There was no impact of the variant on the structure or expression of the Hcn1 protein in the retina. In silico modeling of photoreceptors revealed that the mutated HCN1 channel dramatically reduced light-induced hyperpolarization, resulting in more Ca2+ flux during the response when compared with the wild-type situation. We propose that the light-induced change in glutamate release from photoreceptors during a stimulus will be diminished, significantly blunting the dynamic range of this response. Our data highlight the importance of HCN1 channels to retinal function and suggest that patients with HCN1 pathogenic variants are likely to have a dramatically reduced sensitivity to light and a limited ability to process temporal information.SIGNIFICANCE STATEMENT Pathogenic variants in HCN1 are emerging as an important cause of catastrophic epilepsy. HCN1 channels are ubiquitously expressed throughout the body, including the retina. Electroretinogram recordings from a mouse model of HCN1 genetic epilepsy showed a marked decrease in the photoreceptor sensitivity to light and a reduced ability to respond to high rates of light flicker. No morphologic deficits were noted. Simulation data suggest that the mutated HCN1 channel blunts light-induced hyperpolarization and consequently limits the dynamic range of this response. Our results provide insights into the role HCN1 channels play in retinal function as well as highlighting the need to consider retinal dysfunction in disease caused by HCN1 variants. The characteristic changes in the electroretinogram open the possibility of using this tool as a biomarker for this HCN1 epilepsy variant and to facilitate development of treatments.
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Epilepsia , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Humanos , Masculino , Femenino , Ratones , Animales , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Retina/metabolismo , Electrorretinografía , Epilepsia/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Canales de Potasio/fisiologíaRESUMEN
Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.
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Quimiocina CX3CL1/metabolismo , Retinopatía Diabética/patología , Microglía/fisiología , Retina/patología , Animales , Bencimidazoles/farmacología , Compuestos de Bifenilo/farmacología , Quimiocina CX3CL1/farmacología , Retinopatía Diabética/inducido químicamente , Retinopatía Diabética/metabolismo , Perfilación de la Expresión Génica , Ratones , Microglía/metabolismo , Neuronas/fisiología , Pericitos/patología , Ratas , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/genética , Retina/metabolismo , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacos , Estreptozocina/farmacología , Tetrazoles/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
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Terapia Genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Neovascularización Retiniana/genética , Neovascularización Retiniana/terapia , Animales , Hipoxia de la Célula , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Células HEK293 , Humanos , Inyecciones Intravítreas , Dominios Proteicos , Ratas Sprague-Dawley , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Transgenes , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: To investigate changes in aqueous humor dynamics during intraocular pressure (IOP) elevation induced by circumlimbal suture in mice. METHODS: Ocular hypertension (OHT) was induced by applying a circumlimbal suture behind the limbus in male adult C57BL6/J mice. In the OHT group, the suture was left in place for an average of 8 weeks (n = 10, OHT group). In the sham control group the suture was cut at 2 days (n = 9, sham group) and in the naïve control group (n = 5) no suture was implanted. IOP was measured at baseline across 3 days, 1 h post-suture implantation, and at the chronic endpoint. Anterior segments were assessed using optical coherence tomography (OCT). Episcleral venous pressure (EVP), total outflow facility (C), uveoscleral outflow (Fu) and aqueous humor flow rate (Fin) were determined using a constant-flow infusion model. RESULTS: All aqueous dynamic and chronic IOP outcome measures showed no difference between sham and naïve controls (p > 0.05) and thus these groups were combined into a single control group. IOP was elevated in OHT group compared with controls (p < 0.01). Chronic suture implantation did not change pupil size, anterior chamber depth or iridocorneal angles (p > 0.05). EVP was significantly higher in OHT eyes compared to control eyes (p < 0.01). There was no statistical difference in C, Fu and Fin between groups (p > 0.05). A significant linear correlation was found between IOP and EVP (R2 = 0.35, p = 0.001). CONCLUSIONS: Circumlimbal suture implantation in mouse eyes results in chronic IOP elevation without angle closure. Chronic IOP elevation is likely to reflect higher EVP.
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Humor Acuoso/metabolismo , Presión Intraocular/fisiología , Hipertensión Ocular/fisiopatología , Suturas/efectos adversos , Presión Venosa/fisiología , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Hipertensión Ocular/diagnóstico , Hipertensión Ocular/etiología , Tomografía de Coherencia ÓpticaRESUMEN
Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.
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Oftalmopatías/patología , Oftalmopatías/terapia , Terapia Genética , Neovascularización Patológica/terapia , Animales , Sistemas CRISPR-Cas , Ensayos Clínicos como Asunto , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Oftalmopatías/etiología , Edición Génica , Terapia Genética/métodos , Humanos , Neovascularización Patológica/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset(TM)library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4-10 µmin zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1-2) at micromolar IC50values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.
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Inhibidores de la Angiogénesis , Descubrimiento de Drogas , Fenoles , Quinolinas , Neovascularización Retiniana/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Animales , Animales Modificados Genéticamente , Línea Celular , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Humanos , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Ratones , Fenoles/química , Fenoles/farmacocinética , Fenoles/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Pez CebraRESUMEN
BACKGROUND: Loss of substantia nigra dopaminergic cells and alpha-synuclein (α-syn)-rich intraneuronal deposits within the central nervous system are key hallmarks of Parkinson's disease (PD). Levodopa (L-DOPA) is the current gold-standard treatment for PD. This study aimed to evaluate in vivo retinal changes in a transgenic PD model of α-syn overexpression and the effect of acute levodopa (L-DOPA) treatment. METHODS: Anaesthetised 6-month-old mice expressing human A53T alpha-synuclein (HOM) and wildtype (WT) control littermates were intraperitoneally given 20 mg/kg L-DOPA (50 mg levodopa, 2.5 mg benserazide) or vehicle saline (n = 11-18 per group). In vivo retinal function (dark-adapted full-field ERG) and structure (optical coherence tomography, OCT) were recorded before and after drug treatment for 30 min. Ex vivo immunohistochemistry (IHC) on flat-mounted retina was conducted to assess tyrosine hydroxylase (TH) positive cell counts (n = 7-8 per group). RESULTS: We found that photoreceptor (a-wave) and bipolar cell (b-wave) ERG responses (p < 0.01) in A53T HOM mice treated with L-DOPA grew in amplitude more (47 ± 9%) than WT mice (16 ± 9%) treated with L-DOPA, which was similar to the vehicle group (A53T HOM 25 ± 9%; WT 19 ± 7%). While outer retinal thinning (outer nuclear layer, ONL, and outer plexiform layer, OPL) was confirmed in A53T HOM mice (p < 0.01), L-DOPA did not have an ameliorative effect on retinal layer thickness. These findings were observed in the absence of changes to the number of TH-positive amacrine cells across experiment groups. Acute L-DOPA treatment transiently improves visual dysfunction caused by abnormal alpha-synuclein accumulation. CONCLUSIONS: These findings deepen our understanding of dopamine and alpha-synuclein interactions in the retina and provide a high-throughput preclinical framework, primed for translation, through which novel therapeutic compounds can be objectively screened and assessed for fast-tracking PD drug discovery.
RESUMEN
Retinal hyperspectral imaging (HSI) is a non-invasive in vivo approach that has shown promise in Alzheimer's disease. Parkinson's disease is another neurodegenerative disease where brain pathobiology such as alpha-synuclein and iron overaccumulation have been implicated in the retina. However, it remains unknown whether HSI is altered in in vivo models of Parkinson's disease, whether it differs from healthy aging, and the mechanisms which drive these changes. To address this, we conducted HSI in two mouse models of Parkinson's disease across different ages; an alpha-synuclein overaccumulation model (hA53T transgenic line M83, A53T) and an iron deposition model (Tau knock out, TauKO). In comparison to wild-type littermates the A53T and TauKO mice both demonstrated increased reflectivity at short wavelengths ~ 450 to 600 nm. In contrast, healthy aging in three background strains exhibited the opposite effect, a decreased reflectance in the short wavelength spectrum. We also demonstrate that the Parkinson's hyperspectral signature is similar to that from an Alzheimer's disease model, 5xFAD mice. Multivariate analyses of HSI were significant when plotted against age. Moreover, when alpha-synuclein, iron or retinal nerve fibre layer thickness were added as a cofactor this improved the R2 values of the correlations in certain groups. This study demonstrates an in vivo hyperspectral signature in Parkinson's disease that is consistent in two mouse models and is distinct from healthy aging. There is also a suggestion that factors including retinal deposition of alpha-synuclein and iron may play a role in driving the Parkinson's disease hyperspectral profile and retinal nerve fibre layer thickness in advanced aging. These findings suggest that HSI may be a promising translation tool in Parkinson's disease.
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Modelos Animales de Enfermedad , Envejecimiento Saludable , Imágenes Hiperespectrales , Ratones Transgénicos , Enfermedad de Parkinson , Retina , alfa-Sinucleína , Animales , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/genética , Retina/metabolismo , Retina/diagnóstico por imagen , Retina/patología , Ratones , Envejecimiento Saludable/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Imágenes Hiperespectrales/métodos , Hierro/metabolismo , Humanos , Masculino , Ratones NoqueadosRESUMEN
This study quantified age-related changes to retinal autophagy using the CAG-RFP-EGFP-LC3 autophagy reporter mice and considered how aging impacts autophagic responses to acute intraocular pressure (IOP) stress. IOP was elevated to 50â¯mm Hg for 30â¯minutes in 3-month-old and 12-month-old CAG-RFP-EGFP-LC3 (nâ¯=â¯7 per age group) and Thy1-YFPh transgenic mice (nâ¯=â¯3 per age group). Compared with younger eyes, older eyes showed diminished basal autophagy in the outer retina, while the inner retina was unaffected. Autophagic flux (red:yellow puncta ratio) was elevated in the inner plexiform layer. Three days following IOP elevation, older eyes showed poorer functional recovery, most notably in ganglion cell responses compared to younger eyes (12 months old: -33.4⯱â¯5.3% vs. 3 months mice: -13.4⯱â¯4.5%). This paralleled a reduced capacity to upregulate autophagic puncta volume in the inner retina in older eyes, a response that was seen in younger eyes. Age-related decline in basal and stress-induced autophagy in the retina is associated with greater retinal ganglion cells' susceptibility to IOP elevation.
Asunto(s)
Presión Intraocular , Retina , Ratones , Animales , Células Ganglionares de la Retina/fisiología , Modelos Animales de Enfermedad , Ratones Transgénicos , Autofagia/genéticaRESUMEN
Abnormal alpha-synuclein (α-SYN) protein deposition has long been recognized as one of the pathological hallmarks of Parkinson's disease's (PD). This study considers the potential utility of PD retinal biomarkers by investigating retinal changes in a well characterized PD model of α-SYN overexpression and how these correspond to the presence of retinal α-SYN. Transgenic A53T homozygous (HOM) mice overexpressing human α-SYN and wildtype (WT) control littermates were assessed at 4, 6, and 14 months of age (male and female, n = 15-29 per group). In vivo retinal function (electroretinography, ERG) and structure (optical coherence tomography, OCT) were recorded, and retinal immunohistochemistry and western blot assays were performed to examine retinal α-SYN and tyrosine hydroxylase. Compared to WT controls, A53T mice exhibited reduced light-adapted (cone photoreceptor and bipolar cell amplitude, p < 0.0001) ERG responses and outer retinal thinning (outer plexiform layer, outer nuclear layer, p < 0.0001) which correlated with elevated levels of α-SYN. These retinal signatures provide a high throughput means to study α-SYN induced neurodegeneration and may be useful in vivo endpoints for PD drug discovery.
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Electroretinography and optical coherence tomography imaging allow for non-invasive quantitative assessment of the retina. These approaches have become mainstays for identifying the very earliest impact of hyperglycemia on retinal function and structure in animal models of diabetic eye disease. Moreover, they are essential for assessing the safety and efficacy of novel treatment approaches for diabetic retinopathy. Here, we describe approaches for in vivo electroretinography and optical coherence tomography imaging in rodent models of diabetes.
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Diabetes Mellitus , Retinopatía Diabética , Animales , Electrorretinografía , Tomografía de Coherencia Óptica/métodos , Roedores , Retina/diagnóstico por imagen , Retinopatía Diabética/diagnóstico por imagenRESUMEN
Electroretinography allows for noninvasive functional assessment of the retina and is a mainstay for preclinical studies of retinal function in health and disease. The full-field electroretinogram is useful for a variety of applications as it returns a functional readout from each of the major cell classes within the retina: photoreceptors, bipolar cells, amacrine cells, and retinal ganglion cells. Rodent models are commonly employed in ocular degeneration studies due to the fast throughput of these mammalian species and the conservation of the electroretinogram from the preclinic to the clinic. Here we describe approaches for in vivo electroretinography in rodent models.
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Electrorretinografía , Roedores , Animales , Retina , Células Ganglionares de la Retina , Células AmacrinasRESUMEN
Aging and elevated intraocular pressure (IOP) are two major risk factors for glaucomatous optic neuropathy; a condition characterized by the selective, progressive injury, and subsequent loss of retinal ganglion cells (RGCs). We examined how age modified the capacity for RGCs to functionally recover following a reproducible IOP elevation (50 mmHg for 30 min). We found that RGC functional recovery (measured using electroretinography) was complete by 7 days in 3-month-old mice but was delayed in 12-month-old mice until 14 days. At the 7-day recovery endpoint when RGC function had recovered in young but not older eyes, we examined RGC structural responses to IOP-related stress by analyzing RGC dendritic morphology. ON-RGC cell volume was attenuated following IOP elevation in both young and older mice. We also found that following IOP elevation OFF-RGC dendritic morphology became less complex per cell volume in young mice, an effect that was not observed in older eyes. Our data suggest that adaptations in OFF-RGCs in young eyes were associated with better functional recovery 7 days after IOP elevation. Loss of RGC cellular adaptations may account for delayed functional recovery in older eyes.
RESUMEN
Myelination within the central nervous system (CNS) is crucial for the conduction of action potentials by neurons. Variation in compact myelin morphology and the structure of the paranode are hypothesised to have significant impact on the speed of action potentials. There are, however, limited experimental data investigating the impact of changes in myelin structure upon conductivity in the central nervous system. We have used a genetic model in which myelin thickness is reduced to investigate the effect of myelin alterations upon action potential velocity. A detailed examination of the myelin ultrastructure of mice in which the receptor tyrosine kinase Tyro3 has been deleted showed that, in addition to thinner myelin, these mice have significantly disrupted paranodes. Despite these alterations to myelin and paranodal structure, we did not identify a reduction in conductivity in either the corpus callosum or the optic nerve. Exploration of these results using a mathematical model of neuronal conductivity predicts that the absence of Tyro3 would lead to reduced conductivity in single fibres, but would not affect the compound action potential of multiple myelinated neurons as seen in neuronal tracts. Our data highlight the importance of experimental assessment of conductivity and suggests that simple assessment of structural changes to myelin is a poor predictor of neural functional outcomes.
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Vaina de Mielina , Sustancia Blanca , Potenciales de Acción/fisiología , Animales , Axones/fisiología , Ratones , Vaina de Mielina/ultraestructura , Nervio Óptico/fisiologíaRESUMEN
In addition to well characterized motor symptoms, visual disturbances are increasingly recognized as an early manifestation in Parkinson's disease (PD). A better understanding of the mechanisms underlying these changes would facilitate the development of vision tests which can be used as preclinical biomarkers to support the development of novel therapeutics for PD. This study aims to characterize the retinal phenotype of a mouse model of dopaminergic dysfunction and to examine whether these changes are reversible with levodopa treatment. We use a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD to characterize the neurotoxic effects of MPTP on in vivo retinal function (electroretinography, ERG), retinal structure (optical coherence tomography, OCT) and retinal dopaminergic cell number (tyrosine hydroxylase immunohistochemistry, IHC) at two time points (21 and 45 days) post MPTP model induction. We also investigate the effect of levodopa (L-DOPA) as a proof-of-principle chronic intervention against MPTP-induced changes in the retina. We show that MPTP decreases dopaminergic amacrine cell number (9%, p < 0.05) and that a component of the ERG that involves these cells, in particular oscillatory potential (OP) peak timing, was significantly delayed at Day 45 (7-13%, p < 0.01). This functional deficit was paralleled by outer plexiform layer (OPL) thinning (p < 0.05). L-DOPA treatment ameliorated oscillatory potential deficits (7-13%, p < 0.001) in MPTP animals. Our data suggest that the MPTP toxin slows the timing of inner retinal feedback circuits related to retinal dopaminergic pathways which mirrors findings from humans with PD. It also indicates that the MPTP model causes structural thinning of the outer retinal layer on OCT imaging that is not ameliorated with L-DOPA treatment. Together, these non-invasive measures serve as effective biomarkers for PD diagnosis as well as for quantifying the effect of therapy.
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Intoxicación por MPTP , Enfermedad de Parkinson , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Levodopa/farmacología , Levodopa/uso terapéutico , Intoxicación por MPTP/complicaciones , Intoxicación por MPTP/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Retina/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
There is increasing evidence for the vulnerability of specific retinal ganglion cell (RGC) types in those with glaucoma and in animal models. In addition, the P2X7-receptor (P2X7-R) has been suggested to contribute to RGC death following stimulation and elevated IOP, though its role in RGC dysfunction prior to death has not been examined. Therefore, we examined the effect of an acute, non-ischemic intraocular pressure (IOP) insult (50 mmHg for 30 min) on RGC function in wildtype mice and P2X7-R knockout (P2X7-KO) mice. We examined retinal function using electroretinogram recordings and individual RGC responses using multielectrode arrays, 3 days following acute IOP elevation. Immunohistochemistry was used to examine RGC cell death and P2X7-R expression in several RGC types. Acute intraocular pressure elevation produced pronounced dysfunction in RGCs; whilst other retinal neuronal responses showed lesser changes. Dysfunction at 3 days post-injury was not associated with RGC loss or changes in receptive field size. However, in wildtype animals, OFF-RGCs showed reduced spontaneous and light-elicited activity. In the P2X7-KO, both ON- and OFF-RGC light-elicited responses were reduced. Expression of P2X7-R in wildtype ON-RGC dendrites was higher than in other RGC types. In conclusion, OFF-RGCs were vulnerable to acute IOP elevation and their dysfunction was not rescued by genetic ablation of P2X7-R. Indeed, knockout of P2X7-R also caused ON-RGC dysfunction. These findings aid our understanding of how pressure affects RGC function and suggest treatments targeting the P2X7-R need to be carefully considered.
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Presión Intraocular/fisiología , Receptores Purinérgicos P2X7/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Glaucoma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tonometría Ocular/métodosRESUMEN
Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma. STATEMENT OF SIGNIFICANCE: Recent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.
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Glaucoma , Disco Óptico , Animales , Benzamidas , Modelos Animales de Enfermedad , Glaucoma/tratamiento farmacológico , Presión Intraocular , Ratones , Células Ganglionares de la RetinaRESUMEN
Retinal ganglion cells (RGCs) are the only output neurons of the vertebrate retina, integrating signals from other retinal neurons and transmitting information to the visual centers of the brain. The death of RGCs is a common outcome in many optic neuropathies, such as glaucoma, demyelinating optic neuritis and ischemic optic neuropathy, resulting in visual defects and blindness. There are currently no therapies in clinical use which can prevent RGC death in optic neuropathies; therefore, the identification of new targets for supporting RGC survival is crucial in the development of novel treatments for eye diseases. In this study we identify that the receptor tyrosine kinase, Tyro3, is critical for normal neuronal function in the adult mouse retina. The loss of Tyro3 results in a reduction in photoreceptor and RGC function as measured using electroretinography. The reduction in RGC function was associated with a thinner retinal nerve fiber layer and fewer RGCs. In the central retina, independent of the loss of RGCs, Tyro3 deficiency resulted in a dramatic reduction in the number of RGC dendrites in the inner plexiform layer. Our results show that Tyro3 has a novel, previously unidentified role in retinal function, RGC survival and RGC morphology. The Tyro3 pathway could therefore provide an alternative, targetable pathway for RGC protective therapeutics.
RESUMEN
BACKGROUND: Amyloid-ß soluble oligomers (Aßo) are believed to be the cause of the pathophysiology underlying Alzheimer's disease (AD) and are normally detected some two decades before clinical onset of the disease. Retinal pathology associated with AD pathogenesis has previously been reported, including ganglion cell loss, accumulation of Aß deposits in the retina, and reduction of nerve fiber layer thickness as well as abnormalities of the microvasculature. OBJECTIVE: This study's aim is to better understand the relationship between brain and retinal Aßo deposition and in particular to quantify levels of the toxic Aßo as a function of age in the retina of a rodent model of AD. METHODS: Retinas and brain tissue from 5×FAD mice were stained with Congo red, Thioflavin-T (Th-T), and Aß plaque-specific and Aßo-specific antibodies. RESULTS: We show that retinas displayed an age-dependent increase of Th-T-specific amyloid fibrils. Staining with anti-Aß antibody confirmed the presence of the Aß plaques in all 5×FAD retinas tested. In contrast, staining with anti-Aßo antibody showed an age-dependent decrease of retinal Aßo. Of note, Aßo was observed mainly in the retinal nuclear layers. Finally, we confirmed the localization of Aßo to neurons, typically accumulating in late endosomes, indicating possible impairment of the endocytic pathway. CONCLUSION: Our results demonstrate the presence of intraneuronal Aßo in the retina and its accumulation inversely correlated with retinal Aß plaque deposition, indicating an age-related conversion in this animal model. These results support the development of an early AD diagnostic test targeting Aßo in the eye.
Asunto(s)
Factores de Edad , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Retina/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Placa Amiloide/patología , Retina/patología , Roedores/metabolismoRESUMEN
Purpose: The purpose of this study was to test the hypothesis that the superficial, intermediate, and deep retinal vascular plexus show different responses to intraocular pressure (IOP) elevation. Methods: Anesthetized adult Long Evans rats (n = 14) were imaged using optical coherence tomography angiography (OCTA; Spectralis) at baseline (IOP 10 mm Hg) and in follow-up mode to examine the vasculature during IOP elevation (10 to 110 mm Hg, 10 mm Hg steps, each step 3 minutes). A 20° × 10° field was imaged. Vessel density within a 2D projection image was determined (%) for the superficial vascular complex (SVC), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). Comparisons were made between layers using 2-way repeated measures ANOVA (layer versus IOP) following normalization to baseline (% relative to 10 mm Hg). Results: The three vascular layers responded differently to IOP elevation. For IOPs between 40 and 60 mm Hg, DCP and ICP capillaries were significantly more resistant to IOP elevation than those in the SVC. When IOP was elevated above 70 mm Hg, all layers showed reduced vessel density. IOP induced change in SVC vessel density closely followed reductions in thickness of the inner retinal layers (nerve fiber, ganglion cell, and inner plexiform layer). This close relationship between reductions in tissue thickness and vessel density was less apparent for the ICP and DCP. Conclusions: These data show that the intermediate and deep vascular plexus in the rat retina have a greater capacity for autoregulation against mild IOP elevation but are more affected at high IOP.