RESUMEN
Glaucoma is a multifactorial optic neuropathy that primarily affecting retinal ganglion cells (RGC). Brimonidine is an intraocular pressure-lowering drug with reported neuroprotective properties. This study aimed to compare the neuroprotective effects of topical and intraperitoneal (IP) brimonidine on RGCs from different retinal segments in a murine optic nerve crush (ONC) model. METHODS: forty-one Balb/c mice underwent unilateral ONC and were divided into three study groups: fifteen animals received saline drops twice per day and two additional IP injections of saline; fourteen mice received brimonidine drops twice per day; and 12 mice received brimonidine eye drops twice per day and two additional IP brimonidine injections. Animals were sacrificed seven days post-ONC, and immunohistochemical staining of retinal whole mounts was performed using neuronal NeuN and GFAP staining. Microscopic pictures of the central, middle, and peripheral regions of the retina were taken. The density of the retinal cells was assessed. RESULTS: The total RGC density after ONC and RGC densities in all retinal eccentricities were significantly higher in the brimonidine eye drop and IP combination treatment group than in the saline drop + saline IP, and brimonidine drop treatment groups. CONCLUSIONS: brimonidine eye drops supplemented with IP brimonidine injections improved RGC survival in a preclinical model of ONC.
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Tartrato de Brimonidina , Modelos Animales de Enfermedad , Ratones Endogámicos BALB C , Compresión Nerviosa , Fármacos Neuroprotectores , Traumatismos del Nervio Óptico , Células Ganglionares de la Retina , Animales , Tartrato de Brimonidina/administración & dosificación , Tartrato de Brimonidina/farmacología , Ratones , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/administración & dosificación , Traumatismos del Nervio Óptico/tratamiento farmacológico , Traumatismos del Nervio Óptico/patología , Administración Tópica , Soluciones Oftálmicas , Masculino , Inyecciones IntraperitonealesRESUMEN
Diabetic retinopathy (DR) is a multifactorial disease displaying vascular-associated pathologies, including vascular leakage and neovascularization, ultimately leading to visual impairment. However, animal models accurately reflecting these pathologies are lacking. Vascular endothelial growth factor A (VEGF-A) is an important factor in the development of micro- and macro-vascular pathology in DR. In this study, we evaluated the feasibility of using a cumate-inducible lentivirus (LV) mediated expression of vegf-a to understand DR pathology in vitro and in vivo. Retinal pigment epithelial cells (ARPE-19) were transduced with cumate-inducible LV expressing vegf-a, with subsequent analysis of vegf-a expression and its impact on cell proliferation, viability, motility, and permeability. Cumate tolerability in adult Wistar rat eyes was assessed as an initial step towards a potential DR animal model development, by administering cumate via intravitreal injections (IVT) and evaluating consequent effects by spectral domain optical coherence tomography (SD-OCT), flash electroretinography (fERG), ophthalmic examination (OE), and immunohistochemistry. Transduction of ARPE-19 cells with cumate-inducible LV resulted in ~ 2.5-fold increase in vegf-a mRNA and ~ threefold increase in VEGF-A protein secretion. Transduced cells displayed enhanced cell proliferation, viability, permeability, and migration in tube-like structures. However, IVT cumate injections led to apparent retinal toxicity, manifesting as retinal layer abnormalities, haemorrhage, vitreous opacities, and significant reductions in a- and b-wave amplitudes, along with increased microglial activation and reactive gliosis. In summary, while cumate-inducible LV-mediated vegf-a expression is valuable for in vitro mechanistic studies in cellular drug discovery, its use is not a feasible approach to model DR in in vivo studies due to cumate-induced retinal toxicity.
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Retinopatía Diabética , Lentivirus , Epitelio Pigmentado de la Retina , Factor A de Crecimiento Endotelial Vascular , Animales , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Retinopatía Diabética/patología , Retinopatía Diabética/metabolismo , Lentivirus/genética , Ratas , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Humanos , Ratas Wistar , Proliferación Celular , Modelos Animales de Enfermedad , Línea Celular , Inyecciones Intravítreas , Masculino , Movimiento Celular , Supervivencia Celular , Tomografía de Coherencia Óptica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genéticaRESUMEN
The streptozotocin (STZ)-induced rodent model is one of the most commonly employed models in preclinical drug discovery for diabetic retinopathy (DR). However, standardization and validation of experimental readouts are largely lacking. The aim of this systematic review was to identify and compare the most useful readouts of STZ-induced DR and provide recommendations for future study design based on our findings. We performed a systematic search using 2 major databases, PubMed and EMBASE. Only articles describing STZ-induced DR describing both functional and structural readouts were selected. We also assessed the risk of bias and analyzed qualitative data in the selected studies. We identified 21 studies that met our inclusion/exclusion criteria, using either rats or mice and study periods of 2 to 24 weeks. Glucose level thresholds used to define hyperglycemia were inconsistent between studies, however, most studies used either 250 or 300.6 mg/dL as a defining criterion for hyperglycemia. All included studies performed electroretinography (ERG) and reported a reduction in a-, b-, or c-wave and/or oscillatory potential amplitudes. Spectral-domain optical coherence tomography and fluorescein angiography, as well as immunohistochemical and histopathological analyses showed reductions in retinal thickness, vascular changes, and presence of inflammation. Risk of bias assessment showed that all studies had a high risk of bias due to lack of reporting or correctly following procedures. Our systematic review highlights that ERG represents the most consistent functional readout in the STZ model. However, due to the high risk of bias, caution must be used when interpreting these studies.
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Diabetes Mellitus Experimental , Retinopatía Diabética , Hiperglucemia , Animales , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/patología , Electrorretinografía , Hiperglucemia/patología , Ratones , Ratas , Retina/patología , Roedores , Estreptozocina , Relación Estructura-ActividadRESUMEN
ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5'-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local "purinergic junctions" with CD39low/CD73- neuronal cell bodies and CD39high/CD73- retinal blood vessels. The relevance of the CD73-adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.
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5'-Nucleotidasa/metabolismo , Adenosina/metabolismo , Luz , Retina/efectos de la radiación , 5'-Nucleotidasa/antagonistas & inhibidores , 5'-Nucleotidasa/genética , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Apirasa/genética , Apirasa/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microglía/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Retina/fisiología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismoRESUMEN
Oxidative stress is a known contributor to the progression of dry eye disease pathophysiology, and previous studies have shown that antioxidant intervention is a promising therapeutic approach to reduce the disease burden and slow disease progression. In this study, we evaluated the pharmacological efficacy of the naturally occurring prenylated chalconoid, xanthohumol, in preclinical models for dry eye disease. Xanthohumol acts by promoting the transcription of phase II antioxidant enzymes. In this study, xanthohumol prevented tert-butyl hydroperoxide-induced loss of cell viability in human corneal epithelial (HCE-T) cells in a dose-dependent manner and resulted in a significant increase in expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of phase II endogenous antioxidant enzymes. Xanthohumol-encapsulating poly(lactic-co-glycolic acid) nanoparticles (PLGA NP) were cytoprotective against oxidative stress in vitro, and significantly reduced ocular surface damage and oxidative stress-associated DNA damage in corneal epithelial cells in the mouse desiccating stress/scopolamine model for dry eye disease in vivo. PLGA NP represent a safe and efficacious drug delivery vehicle for hydrophobic small molecules to the ocular surface. Optimization of NP-based antioxidant formulations with the goal to minimize instillation frequency may represent future therapeutic options for dry eye disease and related ocular surface disease.
RESUMEN
INTRODUCTION: To examine the compatibility of intravitreally injected epidermal growth factor (EGF) and amphiregulin as EGF family member. METHODS: Four rabbits (age: 4 months; body weight: 2.5 kg) received three intravitreal injections of EGF (100 ng) uniocularly in monthly intervals and underwent ocular photography, tonometry, biometry, and optical coherence tomography. After sacrificing the rabbits, the globes were histomorphometrically examined. In a second study part, eyes of 22 guinea pigs (age: 2-3 weeks) received two intravitreal administrations of amphiregulin (10 ng) or phosphate buffered solution (PBS) in 10-day interval, or were left untouched. Ten days after the second injection, the guinea pigs were sacrificed, the enucleated eyes underwent histological and immune-histological examinations. RESULTS: The rabbit eyes with EGF injections versus the contralateral untouched eyes did not show significant differences in intraocular pressure (7.5 ± 2.4 mmHg vs. 6.8 ± 2.2 mmHg; P = 0.66), retinal thickness (158 ± 5 µm vs. 158 ± 3 µm; P = 1.0), cell counts in the retinal ganglion cell layer (3.3 ± 1.7 cells/150 µm vs. 3.0 ± 1.4 cells/150 µm; P = 0.83), inner nuclear layer (46.4 ± 23.2 cells/150 µm vs. 39.6 ± 6.4 cells/150 µm; P = 0.61), and outer nuclear layer (215 ± 108 cells/150 µm vs. 202 ± 47 cells/150 µm; P = 0.83), or any apoptotic retinal cells. The guinea pig eyes injected with amphiregulin versus eyes with PBS injections did not differ (P = 0.72) in the degree of microglial activation, and both groups did not differ from untouched eyes in number of apoptotic retinal cells and retinal gliosis. CONCLUSIONS: Intravitreal applications of EGF (100 ng) in rabbits nor intravitreal applications of amphiregulin (10 ng) in guinea pigs led to intraocular specific inflammation or any observed intraocular destructive effect. The findings support the notion of a compatibility of intraocular applied EGF and amphiregulin.
Asunto(s)
Factor de Crecimiento Epidérmico , Retina , Anfirregulina , Animales , Cobayas , Presión Intraocular , Inyecciones Intravítreas , ConejosRESUMEN
One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.
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Modelos Animales de Enfermedad , Isquemia/inducido químicamente , Oxígeno/farmacología , Enfermedades de la Retina/inducido químicamente , Animales , Isquemia/complicaciones , Ratones , Ratones Endogámicos C57BL , Ratas , Retina/efectos de los fármacos , Retina/metabolismo , Retina/fisiopatología , Enfermedades de la Retina/complicaciones , Neovascularización Retiniana/complicacionesRESUMEN
Purpose: Retinal explant cultures provide simplified systems where the functions of the retina and the effects of ocular therapies can be studied in an isolated environment. The purpose of this study was to provide insight into long-term preservation of retinal tissue in culture conditions, enable a deeper understanding of the interdependence of retinal morphology and function, and ensure the reliability of the explant technique for prolonged experiments. Methods: Retinal explants from adult mice were cultured as organotypic culture at the air-medium interface for 14 days in vitro (DIV). Retinal functionality was assessed by multielectrode array technique and morphology by immunohistochemical methods at several time points during culture. Results: Retinal explants retained viability for 14 DIV, although with diminishing neuronal activity, progressing neuronal loss, and increasing reactive gliosis. We recorded spontaneous retinal ganglion cell (RGC) activity up to 14 DIV with temporally changing distribution of RGC firing rates. Light responsiveness was measurable from RGCs for 7 DIV and from photoreceptors for 2 DIV. Apoptotic cells were detected beginning at 3 DIV with their density peaking at 7 DIV. The number of RGCs gradually decreased by 70% during 14 DIV. The change was accompanied by the loss of RGC functionality, resulting in 84% loss of electrically active RGCs. Conclusions: Retinal explants provide a valuable tool for studies of retinal functions and development of ocular therapies. However, critical for long-term use, retinal functionality was lost before structural loss, emphasizing a need for both functional and morphologic readouts to determine the overall state of the cultured retina.
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Electrorretinografía/métodos , Células Ganglionares de la Retina/citología , Conservación de Tejido/métodos , Animales , Supervivencia Celular , Células Cultivadas , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Técnicas de Cultivo de Órganos , Reproducibilidad de los Resultados , Células Ganglionares de la Retina/fisiologíaRESUMEN
PURPOSE: To determine the efficacy of the superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), in vitro in human corneal epithelial (HCE-T) cells and in vivo in a preclinical mouse model for dry-eye disease (DED). METHODS: In vitro, HCE-T cultures were exposed either to tert-butylhydroperoxide (tBHP) to generate oxidative stress or to hyperosmolar conditions modeling cellular stress during DED. Cells were pre-treated with Mn-TM-2-PyP or vehicle. Mn-TM-2-PyP permeability across stratified HCE-T cells was assayed. In vivo, Mn-TM-2-PyP (0.1% w/v in saline) was delivered topically as eye drops in a desiccating stress/scopolamine model for DED. Preclinical efficacy was compared to untreated, vehicle- and ophthalmic cyclosporine emulsion-treated mice. RESULTS: Mn-TM-2-PyP protected HCE-T cells in a dose-dependent manner against tBHP-induced oxidative stress as determined by calculating the IC50 for tBHP in the resazurin, MTT and lactate dehydrogenase release cell viability assays. Mn-TM-2-PyP did not protect HCE-T cells from hyperosmolar insult. Its permeability coefficient across a barrier of HCE-T cells was 1.1⯱â¯0.05â¯×â¯10-6â¯cm/s and the mass balance was 62⯱â¯0.6%. In vivo, topical dosing with Mn-TM-2-PyP resulted in a statistically significant reduction of corneal fluorescein staining, similar to ophthalmic cyclosporine emulsion. Furthermore, Mn-TM-2-PyP significantly reduced leukocyte infiltration into lacrimal glands and prevented degeneration of parenchymal tissue. No protective effect against loss of conjunctival goblet cells was observed. Notably, Mn-TM-2-PyP did not produce ocular toxicity when administered topically. DISCUSSION: Our data suggest that Mn-TM-2-PyP, a prototypic synthetic metalloporphyrin compound with potent catalytic antioxidant activity, can improve signs of DED in vivo by reducing oxidative stress in corneal epithelial cells.
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Síndromes de Ojo Seco/tratamiento farmacológico , Células Caliciformes/patología , Metaloporfirinas/administración & dosificación , Estrés Oxidativo , Animales , Antioxidantes , Recuento de Células , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Células Caliciformes/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Soluciones Oftálmicas/administración & dosificación , Índice de Severidad de la EnfermedadRESUMEN
Purpose: To determine the efficacy of trabodenoson, an adenosine mimetic with highly selective adenosine A1 receptor binding properties, in a preclinical mouse model for dry-eye disease. Methods: Dry-eye disease was induced in adult male C57BL/6 mice using a combination of desiccating environment and transdermal administration of scopolamine. Mice were treated concurrently and twice daily with either vehicle, 6% trabodenoson, or 0.05% cyclosporine (Restasis). Efficacy (P < 0.05 versus vehicle) was determined by clinical assessment of dry-eye symptoms using corneal fluorescein staining and tear volumes and histopathologically by quantifying lacrimal gland pathology and conjunctival goblet cells. Results: Twice-daily topical (ocular) administration of trabodenoson increased tear levels and reduced corneal fluorescein staining (P < 0.05) as compared with vehicle-treated eyes in a mouse model of dry-eye disease. Furthermore, significant infiltration of immune cells in the lacrimal gland and reduced number of mucin-producing conjunctival goblet cells were noted in both untreated and vehicle-treated eyes. Comparatively, trabodenoson treatment significantly reduced lacrimal gland infiltration and increased the number of goblet cells (P < 0.05 for both versus vehicle). These trabodenoson-related effects on lacrimal gland pathology and goblet cells were similar to or better than the effects observed with cyclosporine treatment. Conclusions: Topical ocular delivery of trabodenoson significantly improves the clinical and histopathological signs associated with dry-eye disease in mice. This improvement appears to be related to anti-inflammatory effects from targeting adenosine signaling and represents a novel therapeutic approach to develop for the management of dry-eye disease.
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Modelos Animales de Enfermedad , Síndromes de Ojo Seco/tratamiento farmacológico , Queratoconjuntivitis Seca/tratamiento farmacológico , Nitratos/uso terapéutico , Agonistas del Receptor Purinérgico P1/uso terapéutico , Purinas/uso terapéutico , Administración Oftálmica , Animales , Conjuntiva/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Aparato Lagrimal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Lágrimas/fisiología , Resultado del TratamientoRESUMEN
Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period. The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch's membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch's membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ. This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.
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Neovascularización Coroidal/diagnóstico por imagen , Imagen Multimodal/métodos , Animales , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Humanos , Masculino , RatonesRESUMEN
BACKGROUND: The eye possesses unique anatomical features that make it a valuable target for gene therapy applications. OBJECTIVE: The aim of the current study was to compare transduction efficiency, safety and biodistribution of four viral vectors following intravitreal injection. METHOD: Adenovirus (AdV), Adeno-Associated Virus (AAV), Baculovirus (BV) and Lentivirus (LV) vectors encoding Green Fluorescent Protein (GFP) were injected bilaterally intravitreally into adult C57BL/6OlaHsd mice. Control mice received saline. Eyes and other organs were studied at multiple time points from 3 days to 6 months. Immunohistochemical stainings with retinal cell markers were performed to verify GFP-positive cells. Biodistribution in retina and various non-target tissues was studied using a qPCR method. Inflammatory responses and toxicity were investigated from cryostat eye sections and serum samples. RESULTS: AAV-injected eyes showed GFP expression both in inner and outer retinal cells from 7 days up to 6 months. LV eyes showed long lasting transgene expression mostly in retinal pigment epithelium whereas AdV transiently transduced mainly cells in the anterior chamber. In BV-injected eyes, GFP positivity was very low. qPCR results showed that AdV, AAV and LV spread into the optic nerve, but were below the detection limit in other organs. The strongest immune responses were evoked by intravitreal injections of AdV and BV. The highest concentration of anti-GFP IgG was detected in the AdV-treated group, whereas the AAV group showed the lowest concentration. Neither blood chemistry screen nor the number of apoptotic cells showed any differences between the viral vector and saline injected groups. CONCLUSION: Our findings show that intravitreal gene delivery is safe and feasible with AAV, AdV and lentivirus vectors.
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Adenoviridae/genética , Baculoviridae/genética , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Lentivirus/genética , Epitelio Pigmentado de la Retina/patología , Animales , Células Cultivadas , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuroglía/inmunología , Neuroglía/metabolismo , Neuroglía/patología , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/metabolismo , Distribución TisularRESUMEN
The Finnish variant of late infantile neuronal ceroid lipofuscinosis (CLN5 disease) belongs to a family of neuronal ceroid lipofuscinosis (NCLs) diseases. Vision loss is among the first clinical signs in childhood forms of NCLs. Mutations in CLN5 underlie CLN5 disease. The aim of this study was to characterize how the lack of normal functionality of the CLN5 protein affects the mouse retina. Scotopic electroretinography (ERG) showed a diminished c-wave amplitude in the CLN5 deficient mice already at 1 month of age, indicative of pathological events in the retinal pigmented epithelium. A- and b-waves showed progressive impairment later from 2 and 3 months of age onwards, respectively. Structural and immunohistochemical (IHC) analyses showed preferential damage of photoreceptors, accumulation of autofluorescent storage material, apoptosis of photoreceptors, and strong inflammation in the CLN5 deficient mice retinas. Increased levels of autophagy-associated proteins Beclin-1 and P62, and increased LC3b-II/LC3b-I ratio, were detected by Western blotting from whole retinal extracts. Photopic ERG, visual evoked potentials, IHC and cell counting indicated relatively long surviving cone photoreceptors compared to rods. In conclusion, CLN5 deficient mice develop early vision loss that reflects the condition reported in clinical childhood forms of NCLs. The vision loss in CLN5 deficient mice is primarily caused by photoreceptor degeneration.
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Autofagia , Lipofuscinosis Ceroideas Neuronales/patología , Degeneración Retiniana/patología , Animales , Apoptosis , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Femenino , Fluorescencia , Inflamación/patología , Proteínas de Membrana de los Lisosomas , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/metabolismo , Ratones , Microglía/patología , Lipofuscinosis Ceroideas Neuronales/complicaciones , Lipofuscinosis Ceroideas Neuronales/metabolismo , Degeneración Retiniana/complicaciones , Degeneración Retiniana/metabolismo , Degeneración Retiniana/fisiopatología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Factores de Tiempo , Agudeza VisualRESUMEN
BACKGROUND In glaucoma, non-intraocular pressure (IOP)-related risk factors can result in increased levels of extracellular glutamate, which triggers a cascade of neurodegeneration characterized by the excessive activation of N-methyl-D-aspartate (NMDA). The purpose of our study was to evaluate the glioprotective effects of memantine as a prototypic uncompetitive NMDA blocker on retinal astrocytes in the optic nerve crush (ONC) mouse model for glaucoma. MATERIAL AND METHODS Optic nerve crush was performed on all of the right eyes (n=8), whereas left eyes served as contralateral healthy controls (n=8) in Balb/c/Sca mice. Four randomly assigned mice received 2-µl intravitreal injections of memantine (1 mg/ml) after ONC in the experimental eye. One week after the experiment, optic nerves were dissec-ted and stained with methylene blue. Retinae were detached from the sclera. The tissue was immunostained. Whole-mount retinae were investigated by fluorescent microscopy. Astrocyte counts for each image were performed manually. RESULTS Histological sections of crushed optic nerves showed consistently moderate tissue damage in experimental groups. The mean number of astrocytes per image in the ONC group was significantly lower than in the healthy control group (7.13±1.5 and 10.47±1.9, respectively). Loss of astrocytes in the memantine-treated group was significantly lower (8.83±2.2) than in the ONC group. Assessment of inter-observer reliability showed excellent agreement among observations in control, ONC, and memantine groups. CONCLUSIONS The ONC is an effective method for investigation of astrocytic changes in mouse retina. Intravitreally administered memantine shows a promising glioprotective effect on mouse retinal astrocytes by preserving astrocyte count after ONC.
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Astrocitos/patología , Memantina/administración & dosificación , Memantina/farmacología , Compresión Nerviosa , Neuroglía/patología , Neuroprotección/efectos de los fármacos , Nervio Óptico/patología , Retina/patología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Inyecciones Intravítreas , Ratones Endogámicos BALB C , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Variaciones Dependientes del Observador , Retina/efectos de los fármacos , Retina/metabolismoRESUMEN
The purpose of the study was to devise a fast, reliable and sensitive cell viability assay for assessment of acute cytotoxicity on human corneal epithelial cells by using a clinically relevant exposure time. Acute cytotoxic effects of the pharmaceutical excipients benzalkonium chloride (BAC), macrogolglycerol hydroxystearate (MGHS40), polysorbate 80 (PS80) and marketed ophthalmic formulations (Lumigan(®), Monoprost(®), Taflotan(®), Travatan(®), Xalatan(®)) containing these excipients were tested. Human corneal epithelial cell (HCE-T) viability was assessed by measuring the reduction of resazurin to highly fluorescent resorufin. Expression of the tight junction proteins in HCE-T cells were characterized by immunofluorescence staining. Presence of tight junction proteins in HCE-T cells was demonstrated. BAC preserved ophthalmic formulations showed concentration-dependent and time-dependent cytotoxicity to human corneal epithelium. In contrast, no acute cytotoxicity of non-ionic stabilizing/solubilizing excipients (MGSH40 and PS80) or ophthalmic formulation containing these excipients was observed. Marketed ophthalmic formulations used for glaucoma medication show differential toxicity on human corneal epithelial cells. The present study revealed that BAC-preserved ophthalmic formulations were able to induce acute cytotoxic effects even during a clinically relevant exposure time, which was not observed with MGSH40 and PS80 excipients or ophthalmic formulations containing these excipients.
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Citotoxinas/toxicidad , Composición de Medicamentos , Epitelio Corneal/efectos de los fármacos , Soluciones Oftálmicas/toxicidad , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Córnea/efectos de los fármacos , Córnea/metabolismo , Citotoxinas/administración & dosificación , Relación Dosis-Respuesta a Droga , Composición de Medicamentos/métodos , Epitelio Corneal/metabolismo , Excipientes/toxicidad , Humanos , Masculino , Soluciones Oftálmicas/administración & dosificación , Conservadores Farmacéuticos/administración & dosificación , Conservadores Farmacéuticos/toxicidad , Ratas , Ratas WistarRESUMEN
Huntington's disease (HD) is an inherited neurodegenerative disorder that primarily affects the medium-size GABAergic neurons of striatum. The R6/2 mouse line is one of the most widely used animal models of HD. Previously the hallmarks of HD-related pathology have been detected in photoreceptors and interneurons of R6/2 mouse retina. Here we aimed to explore the survival of retinal ganglion cells (RGCs) and functional integrity of distinct retinal cell populations in R6/2 mice. The pattern electroretinography (PERG) signal was lost at the age of 8 weeks in R6/2 mice in contrast to the situation in wild-type (WT) littermates. This defect may be attributable to a major reduction in photopic ERG responses in R6/2 mice which was more evident in b- than a-wave amplitudes. At the age of 4 weeks R6/2 mice had predominantly the soluble form of mutant huntingtin protein (mHtt) in the RGC layer cells, whereas the aggregated form of mHtt was found in the majority of those cells from the 12-week-old R6/2 mice and onwards. Retinal astrocytes did not contain mHtt deposits. The total numbers of RGC layer cells, retinal astrocytes as well as optic nerve axons did not differ between 18-week-old R6/2 mice and their WT controls. Our data indicate that mHtt deposition does not cause RGC degeneration or retinal astrocyte loss in R6/2 mice even at a late stage of HD-related pathology. However, due to functional deficits in the rod- and cone-pathways, the R6/2 mice suffer progressive deficits in visual capabilities starting as early as 4 weeks; at 8 weeks there is severe impairment. This should be taken into account in any behavioral testing conducted in R6/2 mice.
Asunto(s)
Enfermedad de Huntington/fisiopatología , Retina/fisiopatología , Células Ganglionares de la Retina/metabolismo , Animales , Astrocitos/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Retina/citología , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Células Ganglionares de la Retina/patologíaRESUMEN
Nanocrystal-based drug delivery systems provide important tools for ocular formulation development, especially when considering poorly soluble drugs. The objective of the study was to formulate ophthalmic, intraocular pressure (IOP) reducing, nanocrystal suspensions from a poorly soluble drug, brinzolamide (BRA), using a rapid wet milling technique, and to investigate their IOP reducing effect in vivo. Different stabilizers for the nanocrystals were screened (hydroxypropyl methylcellulose (HPMC), poloxamer F127 and F68, polysorbate 80) and HPMC was found to be the only successful stabilizer. In order to investigate both the effect of an added absorption enhancer (polysorbate 80) and the impact of the free drug in the nanocrystal suspension, formulations in phosphate buffered saline (PBS) at pH 7.4 and pH 4.5 were prepared. Particle size, polydispersity (PI), solid state (DSC), morphology (SEM) as well as dissolution behavior and the uniformity of the formulations were characterized. There was rapid dissolution of BRA (in PBS pH 7.4) from all the nanocrystal formulations; after 1 min 100% of the drug was fully dissolved. The effect was significantly pronounced at pH 4.5, where the dissolved fraction of drug was the highest. The cytotoxicity of nanocrystal formulations to human corneal epithelial cell (HCE-T) viability was tested. The effects of the nanocrystal formulations and the commercial product on the cell viability were comparable. The intraocular pressure (IOP) lowering effect was investigated in vivo using a modern rat ocular hypertensive model and elevated IOP reduction was seen in vivo with all the formulations. Notably, the reduction achieved in experimentally elevated IOP was comparable to that obtained with a marketed product. In conclusion, various BRA nanocrystal formulations, which all showed advantageous dissolution and absorption behavior, were successfully formulated.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/administración & dosificación , Glaucoma/tratamiento farmacológico , Presión Intraocular/efectos de los fármacos , Nanopartículas , Sulfonamidas/administración & dosificación , Tiazinas/administración & dosificación , Administración Oftálmica , Animales , Rastreo Diferencial de Calorimetría , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Modelos Animales de Enfermedad , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Excipientes/química , Glaucoma/enzimología , Glaucoma/fisiopatología , Humanos , Derivados de la Hipromelosa/química , Masculino , Microscopía Electrónica de Rastreo , Nanotecnología , Tamaño de la Partícula , Polisorbatos/química , Ratas Wistar , Solubilidad , Sulfonamidas/química , Sulfonamidas/toxicidad , Propiedades de Superficie , Tecnología Farmacéutica/métodos , Tiazinas/química , Tiazinas/toxicidad , Factores de TiempoRESUMEN
BACKGROUND: Vertebrate genomes undergo epigenetic reprogramming during development and disease. Emerging evidence suggests that DNA methylation plays a key role in cell fate determination in the retina. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina. METHODS: The developing chicken retina and the rd1 and rhodopsin-GFP mouse models of retinal degeneration were used to investigate programmed cell death during retinal development and degeneration. Changes in DNA methylation were determined by immunohistochemistry using antibodies against 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). RESULTS: Punctate patterns of hypermethylation paralleled patterns of caspase3-dependent apoptotic cell death previously reported to occur during development in the chicken retina. Degenerating rd1 mouse retinas, at time points corresponding to the peak of rod cell death, showed elevated signals for 5mC and 5hmC in photoreceptors throughout the retina, with the most intense staining observed in the peripheral retina. Hypermethylation of photoreceptors in rd1 mice was associated with TUNEL and PAR staining and appeared to be cCaspase3-independent. After peak rod degeneration, during the period of cone death, occasional hypermethylation was observed in the outer nuclear layer. CONCLUSION: The finding that cell-specific increases of 5mC and 5hmC immunostaining are associated with the death of retinal neurons during both development and degeneration suggests that changes in DNA methylation may play a role in modulating gene expression during the process of retinal degeneration. During retinal development, hypermethylation of retinal neurons associates with classical caspase-dependent apoptosis as well as caspase-3 independent cell death, while hypermethylation in the rd1 mouse photoreceptors is primarily associated with caspase-3 independent programmed cell death. These findings suggest a previously unrecognized role for epigenetic mechanisms in the onset and/or progression of programed cell death in the retina.
Asunto(s)
Apoptosis , Caspasa 3/metabolismo , Metilación de ADN , Epigénesis Genética , Proteínas del Ojo/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneración Retiniana/metabolismo , Animales , Caspasa 3/genética , Embrión de Pollo , Proteínas del Ojo/genética , Humanos , Ratones , Ratones Transgénicos , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patologíaRESUMEN
PURPOSE: The aim of this study was to characterize the ocular morphology of low-density lipoprotein receptor-deficient apolipoprotein B-100-only mice, where overexpression of insulin-like growth factor II (IGF-II) has been shown to induce glucose intolerance and increase atherosclerotic lesion progression and calcification. METHODS: Fifteen-month-old mice were examined on a normal chow diet and after 3 months of a high-fat Western diet. IGF-II-negative LDLR(-/-)ApoB(100/100) littermates and C57Bl/6J mice served as controls. In vivo color images of the fundi were obtained, and eyes were processed either for retinal flat mounts for assessment of neovascularization or for paraffin-embedded samples for immunohistochemical analyses. RESULTS: IGF-II overexpression and the resulting prediabetic phenotype did not induce microvascular damage when assessed in fundus photographs and retinal whole mounts, and the number of capillaries in IGF-II/LDLR(-/-)ApoB(100/100) mice was not significantly different from LDLR(-/-)ApoB(100/100) mice. However, morphology of the inner nuclear, outer plexiform, and outer nuclear layers was altered in the IGF-II/LDLR(-/-)ApoB(100/100) mice. Moreover, photoreceptor atrophy and thinning of the outer nuclear layer were present. Caspase-3 staining was positive in the photoreceptor inner segment. In addition, retinas of the IGF-II/LDLR(-/-)ApoB(100/100) mice displayed reduced rhodopsin positivity, consistent with the decreased number of photoreceptor cells. CONCLUSIONS: This study reports a novel form of retinopathy with photoreceptor atrophy and abundant changes in retinal morphology in a mouse model of prediabetes and atherosclerosis.
Asunto(s)
Apolipoproteína B-100/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Receptores de LDL/deficiencia , Enfermedades de la Retina/patología , Animales , Apoptosis , Atrofia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patología , Recuento de Células , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Dieta , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores de LDL/metabolismo , Degeneración Retiniana/complicaciones , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Enfermedades de la Retina/complicaciones , Enfermedades de la Retina/metabolismoRESUMEN
Remote regions such as the thalamus undergo secondary degeneration after cerebral ischemia. In rodents, the pathology in the thalamus is characterized by a robust inflammatory reaction, ß-amyloid (Aß) accumulation and calcification. Here we studied whether nonhuman primates subjected to middle cerebral artery occlusion (MCAO) display a similar pathology. Common marmosets (n=4) were subjected to transient MCAO for 3 h. Two sham-operated animals served as controls. All animals underwent MRI examination (T2) on postoperative day 7 to assess the location of the infarct. After a 45-day follow-up period, the animals were perfused for histology to evaluate ß-amyloid and calcium load in the peri-infarct regions and the thalamus. There was no Aß or calcium staining in the sham-operated marmosets. The contralateral hemisphere was devoid of Aß and calcium staining in MCAO animals, except calcium staining in one animal. In the ipsilateral cortex, patchy groups of Aß-positive cells were observed. Occasional calcium staining was observed in the peri-infarct regions, lesion core, and remote regions such as the substantia nigra. The most important, the thalamus was devoid of any sign of Aß and calcium aggregation in MCAO animals. Staining for glial fibrillary acidic protein (GFAP) showed marked astrogliosis in the ipsilateral cortex and thalamus. In conclusion, our preliminary study in marmosets did not identify Aß and calcium pathology in the thalamus following cerebral ischemia as shown in rodents.