RESUMEN
An insult can trigger a protective response or even cell death depending on different factors that include the duration and magnitude of the event and the ability of the cell to activate protective intracellular signals, including inflammatory cytokines. Our previous work showed that the treatment of Lister Hooded rat retinal cell cultures with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, increases the survival of retinal ganglion cells (RGCs) kept in culture for 48 h after axotomy. Here we aim to analyze how PMA modulates the levels of TNF-α and IL-1ß (both key inflammatory mediators) and the impact of this modulation on RGCs survival. We hypothesize that the increase in RGCs survival mediated by PMA treatment depends upon modulation of the levels of IL-1ß and TNF-α. The effect of PMA treatment was assayed on cell viability, caspase 3 activation, TNF-α and IL-1ß release and TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) levels. PMA treatment increases IL-1ß and TNF-α levels in 15 min in culture and increases the release of both cytokines after 30 min and 24 h, respectively. Both IL-1ß and TNF-α levels decrease after 48 h of PMA treatment. PMA treatment also induces an increase in TNFRII levels while decreasing TNFRI after 24 h. PMA also inhibited caspase-3 activation, and decreased ROS production and EthD-1/calcein ratio in retinal cell cultures leading to an increase in cell viability. The neutralization of IL-1ß (anti-IL1ß 0,1ng/mL), the neutralization of TNF-α (anti-TNF-α 0,1ng/mL) and the TNF-α inhibition using a recombinant soluble TNFRII abolished PMA effect on RGCs survival. These data suggest that PMA treatment induces IL1ß and TNF-α release and modulation of TNFRI/TNFRII expression promoting RGCs survival after axotomy.
Asunto(s)
Proteína Quinasa C/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Animales Recién Nacidos , Axotomía/efectos adversos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Interleucina-1beta/metabolismo , Masculino , Cultivo Primario de Células , Ratas , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Células Ganglionares de la Retina/metabolismo , Inhibidores del Factor de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Interleukin-2 (IL-2) is a classical pro-inflammatory cytokine known to display neuroprotective roles in the central nervous system including the retina. In the present study, we investigate the molecular targets involved in the neurotrophic effect of IL-2 on retinal ganglion cells (RGC) after optic nerve axotomy. Analysis of retrograde labeling of RGC showed that common cell survival mediators, as Trk receptors, Src, PI3K, PKC, and intracellular calcium do not mediate the neurotrophic effect of IL-2 on RGC. No involvement of MAPK p38 was also observed. However, other MAPKs as MEK and JNK appear to be mediating this IL-2 effect. Our data also indicate that JAK2/3 are important intracellular proteins for the IL-2 effect. Interestingly, we demonstrate that the IL-2 effect depends on dopamine D1 receptors (D1R), the cAMP/PKA pathway, interleukin-10 (IL-10), and NF-κB, suggesting that RGC survival induced by IL-2 encompasses a molecular network of major complexity. In addition, treatment of retinal cells with recombinant IL-10 or 6-Cl-pb (D1R full agonist) was able to increase RGC survival similar to IL-2. Taken together, our results suggest that after optic nerve axotomy, the increase in RGC survival triggered by IL-2 is mediated by IL-10 and D1R along with the intracellular pathways of MAPKs, JAK/STAT, and cAMP/PKA.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Interleucina-10/metabolismo , Interleucina-2/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Animales Recién Nacidos , Axotomía , Células Cultivadas , Femenino , Masculino , FN-kappa B/metabolismo , Factores de Crecimiento Nervioso/farmacología , Nervio Óptico/cirugía , Ratas , Células Ganglionares de la Retina/metabolismoRESUMEN
Glaucoma is a blindness-causing disease that involves selective damage to retinal ganglion cells (RGCs) and their axons. A subset of RGCs expressing the photopigment melanopsin regulates non-image-forming visual system functions, such as pupillary light reflex and circadian rhythms. We analyzed the effect of melatonin on the non-image-forming visual system alterations induced by experimental glaucoma. For this purpose, male Wistar rats were weekly injected with vehicle or chondroitin sulfate into the eye anterior chamber. The non-image-forming visual system was analyzed in terms of (1) melanopsin-expressing RGC number, (2) anterograde transport from the retina to the olivary pretectal nucleus and the suprachiasmatic nuclei, (3) blue- and white light-induced pupillary light reflex, (4) light-induced c-Fos expression in the suprachiasmatic nuclei, (5) daily rhythm of locomotor activity, and (6) mitochondria in melanopsin-expressing RGC cells. Melatonin prevented the effect of experimental glaucoma on melanopsin-expressing RGC number, blue- and white light-induced pupil constriction, retina-olivary pretectal nucleus, and retina- suprachiasmatic nuclei communication, light-induced c-Fos expression in the suprachiasmatic nuclei, and alterations in the locomotor activity daily rhythm. In addition, melatonin prevented the effect of glaucoma on melanopsin-expressing RGC mitochondrial alterations. These results support that melatonin protected the non-image-forming visual system against glaucoma, probably through a mitochondrial protective mechanism.
Asunto(s)
Antioxidantes/administración & dosificación , Glaucoma/prevención & control , Melatonina/administración & dosificación , Células Ganglionares de la Retina/efectos de los fármacos , Visión Ocular/efectos de los fármacos , Animales , Glaucoma/inducido químicamente , Glaucoma/metabolismo , Luz/efectos adversos , Masculino , Ratas , Ratas Wistar , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/metabolismo , Núcleo Supraquiasmático/efectos de los fármacos , Núcleo Supraquiasmático/metabolismo , Visión Ocular/fisiologíaRESUMEN
Müller glial cells, the major glial cell type in the retina, are activated by most retina injuries, leading to an increased proliferation and migration that contributes to visual dysfunction. The molecular cues involved in these processes are still ill defined. We demonstrated that sphingosine-1-phosphate (S1P), a bioactive sphingolipid, promotes glial migration. We now investigated whether ceramide-1-phosphate (C1P), also a bioactive sphingolipid, was involved in Müller glial cell migration. We evaluated cell migration in primary Müller glial cultures, prepared from newborn rat retinas, by the scratch wound assay. Addition of either 10 µM C8-ceramide-1-phosphate (C8-C1P) or 5 µM C16-C1P (a long chain, natural C1P) stimulated glial migration. Inhibiting PI3K almost completely blocked C8-C1P-elicited migration whereas inhibition of ERK1-2/MAPK pathway diminished it and p38MAPK inhibition did not affect it. Pre-treatment with a cytoplasmic phospholipase A2 (cPLA2) inhibitor markedly reduced C8-C1P-induced migration. Inhibiting ceramide kinase (CerK), the enzyme catalyzing C1P synthesis, partially decreased glial migration. Combined addition of S1P and C8-C1P promoted glial migration to the same extent as when they were added separately, suggesting they converge on their downstream signaling to stimulate Müller glia migration. These results suggest that C1P addition stimulated migration of glial Müller cells, promoting the activation of cPLA2, and the PI3K and ERK/MAPK pathways. They also suggest that CerK-dependent C1P synthesis was one of the factors contributing to glial migration, thus uncovering a novel role for C1P in controlling glial motility.
Asunto(s)
Ceramidas/farmacología , Células Ependimogliales/citología , Células Ganglionares de la Retina/citología , Animales , Animales Recién Nacidos , Movimiento Celular/efectos de los fármacos , Células Ependimogliales/efectos de los fármacos , Modelos Animales , Ratas , Ratas Wistar , Células Ganglionares de la Retina/efectos de los fármacos , Transducción de SeñalRESUMEN
We are reporting for the first time the synthesis and application of an innovative nanometric system for the controlled topic release of melatonin in the retina. The ethylcellulose nanocapsules were characterized by diverse physicochemical techniques (scanning electron microscopy, zeta potential, hydrodynamic diameters) and an in vitro release study was done. A complete ex vivo and in vivo trans-corneal permeation and an irritation study were carried out with the new formulations in albino rabbits, to which a retinal degenerative model was induced. The results obtained demonstrate that the in vitro release of melatonin (1 mg/mL and 2 mg/mL) transported by nanocapsules is slower when compared to a solution of melatonin. Greater penetration of melatonin through the cornea was demonstrated by ex vivo and in vivo tests. This can be attributable to an enhanced neuroprotective effect of melatonin on retinal ganglion cells when it is included in ethylcellulose nanocapsules compared to a solution of melatonin. These outstanding findings add promising new perspectives to current knowledge about administrations using nano-technological tools in the treatment of neurodegenerative diseases at the ocular level.
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Celulosa/análogos & derivados , Melatonina/administración & dosificación , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/patología , Animales , Antioxidantes/administración & dosificación , Celulosa/farmacología , Modelos Animales de Enfermedad , Composición de Medicamentos , Microscopía Electrónica de Rastreo , Nanopartículas/ultraestructura , Conejos , Degeneración Retiniana/diagnóstico , Células Ganglionares de la Retina/efectos de los fármacosRESUMEN
Diabetes produces several changes in the body triggered by high glycemia. Some of these changes include altered metabolism, structural changes in blood vessels and chronic inflammation. The eye and particularly the retinal ganglion cells (RGCs) are not spared, and the changes eventually lead to cell loss and visual function impairment. Understanding the mechanisms resulting in RGC damage and loss from diabetic retinopathy is essential to find an effective treatment. This review focuses mainly on the signaling pathways and molecules involved in RGC loss and the potential therapeutic approaches for the prevention of this cell death. Throughout the manuscript it became evident that multiple factors of different kind are responsible for RGC damage. This shows that new therapeutic agents targeting several factors at the same time are needed. Alpha-1 antitrypsin as an anti-inflammatory agent may become a suitable option for the treatment of RGC loss because of its beneficial interaction with several signaling pathways involved in RGC injury and inflammation. In conclusion, alpha-1 antitrypsin may become a potential therapeutic agent for the treatment of RGC loss and processes behind diabetic retinopathy.
Asunto(s)
Diabetes Mellitus/patología , Retinopatía Diabética/patología , Células Ganglionares de la Retina/patología , Animales , Muerte Celular , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Terapia Molecular Dirigida , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The aim of the present report was to analyze the involvement of glutamate neurotoxicity in retinal ganglion cell loss and optic nerve damage induced by experimental optic neuritis. For this purpose, the authors used an optic neuritis model induced by immunisation with myelin oligodendrocyte glycoprotein (AON). The authors describe a correlation in the timing of retinal ganglion cell (RGC) loss with alterations in the optic nerve actin cytoskeleton dynamic, and visual dysfunction. In addition, they show that an intravitreal injection of glutamate mimics, and an NMDA receptor antagonist avoids the effect of pre-clinical AON on visual functions and RGC number, as well as on optic nerve actin cytoskeleton. Taken together, their results support that avoiding glutamate neurotoxicity could become a new therapeutic approach for optic neuritis treatment.
Asunto(s)
Neuritis Óptica/inmunología , Neuritis Óptica/metabolismo , Receptores de N-Metil-D-Aspartato/inmunología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Humanos , Glicoproteína Mielina-Oligodendrócito/toxicidad , Neuritis Óptica/inducido químicamente , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/inmunología , Células Ganglionares de la Retina/metabolismoRESUMEN
Enriched environment (EE) protects the retina from adult rats against ischemia/reperfusion (I/R) injury; however, how the components of EE contribute to the recovery after retinal ischemic damage remains unclear. We analyzed the contribution of social, cognitive, and visual stimulation on functional and histological alterations induced by I/R. Male Wistar rats were submitted to unilateral ischemia by increasing intraocular pressure to 120 mmHg for 40 min. After ischemia, animals were housed in the following conditions: standard environment (SE), enriched environment (EE), novelty environment (NE), standard social environment (SoE), standard visual environment (SVE), or visual environment (VE). In another set of experiments, rats were submitted to bilateral ischemia and housed in SE or EE. At 2 weeks post-ischemia, rats were subjected to electroretinography and histological analysis. EE (but not SoE or NE) afforded functional and histological protection against unilateral ischemia. EE did not induce protection in animals submitted to bilateral ischemia. VE protected retinal function and histology and increased retinal BDNF levels, while a TrkB receptor antagonist prevented the protective effect of VE against I/R damage. In animals submitted to unilateral ischemia, EE and VE induced an increase in c-fos immunoreactivity in the ipsi and contralateral superior colliculus, whereas in animals submitted to bilateral ischemia, no changes in c-fos-immunoreactivity were observed in either superior colliculus from EE-housed animals. These results support that visual stimulation could be a potent stimulus for driving retinal protection in adult rats through a BDNF/TrkB-dependent mechanism, likely involving the superior colliculus.
Asunto(s)
Isquemia/patología , Estimulación Luminosa , Retina/patología , Retina/efectos de la radiación , Animales , Azepinas/farmacología , Benzamidas/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Electrorretinografía , Locomoción/efectos de los fármacos , Masculino , Ratas Wistar , Retina/efectos de los fármacos , Retina/fisiopatología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/efectos de la radiaciónRESUMEN
After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. Overexpression of PEDF by intravitreal delivery of AAV2 vector significantly increased Tuj1-positive cells survival and modulated FGF-2, IL-1ß, Iba-1, and GFAP immunostaining in the ganglion cell layer (GCL) at 4 weeks after optic nerve crush, although it could not promote axonal outgrowth. The combination of AAV2.PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.
Asunto(s)
Proteínas del Ojo/farmacología , Factores de Crecimiento Nervioso/farmacología , Traumatismos del Nervio Óptico/terapia , Células Ganglionares de la Retina/efectos de los fármacos , Serpinas/farmacología , Animales , Axones/fisiología , Línea Celular Tumoral , Supervivencia Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Modelos Animales de Enfermedad , Proteínas del Ojo/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Compresión Nerviosa , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Neuroprotección , Nervio Óptico , Ratas Wistar , Retina , Células Ganglionares de la Retina/metabolismo , Serpinas/metabolismoRESUMEN
PURPOSE: The current study was undertaken to investigate whether Brazilian green propolis (BGP) can increase the viability of retinal ganglion cells (RGCs) in ischemic mouse retina, and examined the possible mechanisms underlying this neuroprotection. MATERIALS AND METHODS: C57BL/6J mice were subjected to constant elevation of intraocular pressure for 60 min to establish retinal ischemia-reperfusion injury. Mice then received saline or BGP (200 mg/kg) intraperitoneally once daily until sacrifice. The expression of hypoxia-inducing factor (HIF)-1α and glial fibrillary acidic protein (GFAP) and the level of histone acetylation were assessed at 1, 3, and 7 days after injury. The expression of Bax, Bcl-2, p53, NF-κB, Nrf2, and HO-1 were also analyzed at 3 days after injury. The neuroprotective effect of BGP treatment on RGC survival was evaluated using Brn3a immunohistochemical staining. RESULTS: The expression of HIF-1α and GFAP was increased and the level of histone acetylation decreased in saline-treated ischemic retinas within 7 days. BGP treatment effectively attenuated the elevated expression of HIF-1α, GFAP, Bax, NF-κB and p53. The expression of Bcl-2, Nrf2, HO-1 and the level of histone acetylation increased by BGP treatment, resulting in a significant difference between BGP-treated and saline-treated retinas. Immunohistochemical staining for Brn3a also revealed that BGP treatment protected against RGC loss in ischemic retina. CONCLUSIONS: Our results suggest that BGP has a neuroprotective effect on RGCs through the upregulation of histone acetylation, downregulation of apoptotic stimuli, and suppression of NF-κB mediated inflammatory pathway in ischemic retina. These findings suggest that BGP is a potential neuroprotective agent against RGC loss under oxidative stress.
Asunto(s)
Fármacos Neuroprotectores/uso terapéutico , Própolis/uso terapéutico , Daño por Reperfusión/tratamiento farmacológico , Enfermedades de la Retina/tratamiento farmacológico , Células Ganglionares de la Retina/efectos de los fármacos , Acetilación , Animales , Brasil , Supervivencia Celular , Proteína Ácida Fibrilar de la Glía/metabolismo , Histonas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/química , Estrés Oxidativo , Própolis/química , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Factor de Transcripción Brn-3A/metabolismo , Regulación hacia ArribaRESUMEN
Oxidative stress triggers ocular neurodegenerative diseases, such as glaucoma or macular degeneration. The increase of reactive oxygen and nitrogen species in retinal ganglion cells (RGCs) causes damage to the structure and function of the axons that make up the optic nerve, leading to cell death arising from apoptosis, necrosis or autophagy in the RCGs. The use of antioxidants to prevent visual neurodegenerative pathologies is a novel and possibly valuable therapeutic strategy. To investigate in vitro and in vivo neuroprotective efficacy of melatonin (MEL) in RGCs, we used a model of oxidative glutamate (GLUT) toxicity in combination with l-butionin-S, R-sulfoximine (BSO), which induces cell death by apoptosis through cytotoxicity and oxidative stress mechanisms. Histological sectioning and immunohistochemical assays using the TUNEL technique were performed to determine the damage generated in affected cells and to observe the death process of RGCs. Whit BSO-GLUT the results revealed a progressive RGCs death without any significant evidence of a decreased retinal function after 9â¯days of treatment. In this way, we were able to develop a retinal degeneration model in vivo to carry out treatment with MEL and observed an increase in the survival percentage of RGCs, showing that BSO-GLUT could not exert an oxidant effect on cells to counteract the effect of MEL. These findings reveal that MEL has a neuroprotective and antiapoptotic effect as evidenced by the reduction of oxidative stress damage. MEL demonstrated in this model makes it a promising neuroprotective agent for the treatment of ocular neurodegenerative diseases when administered locally.
Asunto(s)
Melatonina/farmacología , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Embrión de Pollo , Ácido Glutámico/farmacología , Técnicas In Vitro , Estrés Oxidativo/efectos de los fármacos , Conejos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismoRESUMEN
Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48â¯h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50â¯ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Agonistas Muscarínicos/farmacología , Factor de Crecimiento Nervioso/genética , Proteína Quinasa C-delta/genética , Receptor Muscarínico M1/genética , Células Ganglionares de la Retina/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Animales , Animales Recién Nacidos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica , Antagonistas Muscarínicos/farmacología , Factor de Crecimiento Nervioso/metabolismo , Pirenzepina/análogos & derivados , Pirenzepina/farmacología , Cultivo Primario de Células , Proteína Quinasa C-delta/metabolismo , Ratas , Receptor Muscarínico M1/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVES: Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model. METHODS: Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy. RESULTS: Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas. CONCLUSIONS: The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.
Asunto(s)
Eritropoyetina/farmacología , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Traumatismos de las Arterias Carótidas/cirugía , Arteria Carótida Común/cirugía , Recuento de Células , Modelos Animales de Enfermedad , Ectodisplasinas/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/farmacología , Masculino , Ratas Wistar , Enfermedades de la Retina/patologíaRESUMEN
OBJECTIVES: Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model. METHODS: Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy. RESULTS: Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas. CONCLUSIONS: The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.
Asunto(s)
Animales , Masculino , Células Ganglionares de la Retina/efectos de los fármacos , Eritropoyetina/farmacología , Fármacos Neuroprotectores/farmacología , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Enfermedades de la Retina/patología , Recuento de Células , Factores de Crecimiento de Célula Hematopoyética/farmacología , Ratas Wistar , Arteria Carótida Común/cirugía , Traumatismos de las Arterias Carótidas/cirugía , Modelos Animales de Enfermedad , Ectodisplasinas/efectos de los fármacosRESUMEN
The aim of this study was to better understand the role of apoptosis in a retinal ischaemia-reperfusion injury model and to determine whether sildenafil citrate treatment can prevent retinal cell apoptosis. Thirty-six rats were divided into a control group (n = 6) and two experimentally induced ischaemia-reperfusion groups (7 and 21 days; n = 15 per group). The induced ischaemia-reperfusion groups were treated with sildenafil for 7 and 21 days (n = 10 per group), and 10 animals were treated with a placebo for the same period (n = 5 per group). Paracentesis of the anterior chamber was performed with a 30-G needle attached to a saline solution (0.9%) bag positioned at a height of 150 cm above the eye for 60 min. Intraocular pressure was measured by rebound tonometer (TonoVet® ). The eyes were analysed by histology and morphometry, and by immunohistochemistry and qRT-PCR for expression of Caspase-7, Caspase-6, Caspase-9, Tnf-r2, Fas-l, Bcl-2 and Bax. Sildenafil-treated animals showed lower levels of histopathological changes (inflammatory, cellular and tissue) than their placebo-treated counterparts at both 7 and 21 days. The retinal ganglion cell layer (RGC) was preserved in the sildenafil groups (SG), with a cell count closer to control than in the placebo groups (PG). Caspase-7 expression was significantly higher in both treated groups at 7 days compared to controls. Gene expression levels in both treatment groups differed from the controls, but in SG Bax and Caspase-6 expression levels were similar to control animals. These results suggest that the main mechanism of retinal cell death in this model is apoptosis, as there is an increase in pro-apoptotic factors and decrease in the anti-apoptotic ones. Also, sildenafil seems to protect the retinal ganglion cell layer from apoptosis. Cell survival was evident in the histological and morphometric analyses, and sildenafil treatment had a protective effect in the apoptosis pathways, with gene expression levels in SG similar to the controls.
Asunto(s)
Daño por Reperfusión/prevención & control , Enfermedades de la Retina/prevención & control , Vasos Retinianos/patología , Citrato de Sildenafil/uso terapéutico , Vasodilatadores/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Regulación de la Expresión Génica/efectos de los fármacos , Presión Intraocular/efectos de los fármacos , Masculino , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Ratas Endogámicas Lew , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Enfermedades de la Retina/fisiopatología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patologíaRESUMEN
Diabetic retinopathy is a severe retinal complication that diabetic patients are susceptible to present. Although this disease is currently characterized as a microvascular disease, there is growing evidence that neural changes occur and maybe precede vascular impairments. Using chicken retina, an avascular tissue with no direct contact with blood vessels and neural retina, this study aimed to evaluate the influence of acute exposure to high glucose concentration in the retinal GABAergic system, and the role of nitric oxide (NO) in this modulation. Therefore, in ex vivo experiments, retinas were incubated in control (10 mM glucose) or high glucose condition (35 mM) for 30 min. By using DAF-FM to evaluate NO production, it was possible to show that high glucose (HG) significantly increased NO levels in the outer nuclear layer, inner nuclear layer (outer and inner portion), and inner plexiform layer. It was also observed that HG increased GABA immunoreactivity (IR) in amacrine and horizontal cells. HG did not change glutamic acid decarboxylase-IR, whereas it decreased GABA Transporter (GAT) 1-IR and increased GAT-3-IR. The co-treatment with 7-NI, an inhibitor of neuronal nitric oxide synthase (nNOS), blocked all changes stimulated by HG exposure. The concomitant exposure with SNAP-5114, a GAT-2/3 inhibitor, blocked the increase in GABA-IR caused by HG incubation. Therefore, our data suggest that hyperglycemia induces GABA accumulation in the cytosol by modulating GABA transporters. This response is dependent on NO production and signaling.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética/metabolismo , Glucosa/administración & dosificación , Óxido Nítrico/biosíntesis , Células Ganglionares de la Retina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Pollos , Retinopatía Diabética/fisiopatología , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patologíaRESUMEN
Ouabain is a steroid hormone that binds to the enzyme Na(+), K(+) - ATPase and stimulates different intracellular pathways controlling growth, proliferation and cell survival. IL-1ß and TNF-α are pleiotropic molecules, conventionally regarded as pro-inflammatory cytokines with well-known effects in the immune system. In addition, IL-1ß and TNF-α also play important roles in the nervous system including neuroprotective effects. Previous data from our group showed that ouabain treatment is able to induce an increase in retinal ganglion cell survival kept in mixed retinal cell cultures. The aim of this work was to investigate if IL-1ß and TNF-α could be mediating the trophic effect of ouabain on retinal ganglion cells. Our results show that the trophic effect of ouabain on retinal ganglion cell was inhibited by either anti-IL-1ß or anti-TNF-α antibodies. In agreement, IL-1ß or TNF-α increased the retinal ganglion cells survival in a dose-dependent manner. Accordingly, ouabain treatment induces a temporal release of TNF-α and IL-1ß from retinal cell cultures. Interestingly, TNF-α and IL-1ß regulate each other intracellular levels. Our results suggest that ouabain treatment triggers the activation of TNF-α and IL-1ß signaling pathways leading to an increase in retinal ganglion cell survival.
Asunto(s)
Supervivencia Celular/inmunología , Interleucina-1beta/inmunología , Ouabaína/administración & dosificación , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Mediadores de Inflamación/inmunología , Ratas , Células Ganglionares de la Retina/patologíaRESUMEN
After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.
Asunto(s)
Axones/efectos de los fármacos , Glicoproteínas/farmacología , Traumatismos del Nervio Óptico/tratamiento farmacológico , Polivinilos/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Degeneración Walleriana/tratamiento farmacológico , Animales , Axones/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Compresión Nerviosa , Traumatismos del Nervio Óptico/patología , Ratas Wistar , Células Ganglionares de la Retina/patología , Factores de Tiempo , Resultado del Tratamiento , Degeneración Walleriana/patologíaRESUMEN
OBJECTIVE: To investigate whether being anesthesia administered at least once in early life influenced 3 main proxies of visual function: visual acuity, refractive error, and optic nerve health in young adulthood. STUDY DESIGN: At age 20 years, participants of the Western Australian Pregnancy Cohort Study had comprehensive ocular examinations including visual acuity, postcycloplegic refraction, and multiple scans of the optic disc. We identified individuals who had at least 1 procedure requiring anesthesia during the first 3 years of life (between 1990 and 1994) and compared their visual outcomes with nonexposed individuals. We excluded 40 participants with strabismus or other ophthalmic disease or surgery and 136 with non-European background. RESULTS: Of 834 participants, 15.2% (n = 127) were exposed to anesthesia at least once before age 3 years. In both exposed and nonexposed groups, median visual acuity (measured using the logarithm of the minimum angle of resolution [LogMAR] chart) was -0.06 LogMAR in the right eye and -0.08 LogMAR in the left eye (P > .05). Median spherical equivalent refractive error was +0.44 diopters (IQR -0.25, +0.63) and +0.31 diopters (IQR -0.38, +0.63) in the exposed and nonexposed group, respectively (P = .126). No difference was detected in mean global retinal nerve fiber layer thickness of the 2 groups (100.7 vs 100.1 µm, P = .830). CONCLUSIONS: We were unable to demonstrate an association of exposure to anesthesia as a child with reduced visual acuity or increased myopia or thinning of retinal nerve fiber layer. These findings support the view that anesthesia is unlikely to impair visual development, but further work is needed to establish whether more subtle defects are present and repeated exposures have any effects.
Asunto(s)
Anestesia/efectos adversos , Miopía/inducido químicamente , Fibras Nerviosas/efectos de los fármacos , Células Ganglionares de la Retina/efectos de los fármacos , Agudeza Visual/efectos de los fármacos , Adolescente , Australia , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Masculino , Miopía/patología , Fibras Nerviosas/patología , Embarazo , Estudios Prospectivos , Células Ganglionares de la Retina/patología , Adulto JovenRESUMEN
After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.