RESUMEN
Purpose: Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods: Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results: In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions: Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Regulación de la Expresión Génica , Terapia Genética/métodos , Glaucoma/complicaciones , Regeneración Nerviosa/genética , Enfermedades Neurodegenerativas/terapia , Neuroprotección/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Muerte Celular , Modelos Animales de Enfermedad , Femenino , Glaucoma/genética , Glaucoma/patología , Masculino , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/genética , Ratas , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.
Asunto(s)
Sensibilidad de Contraste/fisiología , Eliminación de Gen , Retina/enzimología , Sialiltransferasas/deficiencia , Sialiltransferasas/genética , Agudeza Visual/fisiología , Animales , Electrorretinografía/métodos , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Estimulación Luminosa/métodosRESUMEN
Glaucoma is a neurodegenerative disorder characterized by the progressive functional impairment and degeneration of the retinal ganglion cells (RGCs) and their axons, and is the leading cause of irreversible blindness worldwide. Current management of glaucoma is based on reduction of high intraocular pressure (IOP), one of its most consistent risk factors, but the disease proceeds in almost half of the patients despite such treatments. Several experimental models of glaucoma have been developed in rodents, most of which present shortcomings such as high surgical invasiveness, slow learning curves, damage to the transparency of the optic media which prevents adequate functional assessment, and variable results. Here we describe a novel and simple method to induce ocular hypertension in pigmented rats, based on low-temperature cauterization of the whole circumference of the limbal vascular plexus, a major component of aqueous humor drainage and easily accessible for surgical procedures. This simple, low-cost and efficient method produced a reproducible subacute ocular hypertension with full clinical recovery, followed by a steady loss of retinal ganglion cells and optic axons, accompanied by functional changes detected both by electrophysiological and behavioral methods.
Asunto(s)
Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Glaucoma/etiología , Glaucoma/metabolismo , Animales , Biomarcadores , Muerte Celular , Electrorretinografía , Técnica del Anticuerpo Fluorescente , Glaucoma/diagnóstico , Inmunohistoquímica , Presión Intraocular , Degeneración Nerviosa , Desempeño Psicomotor , Ratas , Retina/metabolismo , Retina/patología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
BACKGROUND: Mesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton's jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer's disease-linked amyloid beta oligomers (AßOs). METHODS: We isolated and characterized EVs released by human Wharton's jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AßOs. RESULTS: hMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AßOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AßOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole. CONCLUSIONS: hMSC-EVs protected hippocampal neurons from damage induced by AßOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/citología , Neuronas/patología , Neuroprotección , Estrés Oxidativo , Sinapsis/patología , Gelatina de Wharton/citología , Animales , Biomarcadores/metabolismo , Catalasa/metabolismo , Exosomas/metabolismo , Exosomas/ultraestructura , Vesículas Extracelulares/efectos de los fármacos , Vesículas Extracelulares/ultraestructura , Hipocampo/patología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Multimerización de Proteína , Ratas , Especies Reactivas de Oxígeno/metabolismo , Sinapsis/efectos de los fármacosRESUMEN
Diabetic retinopathy is the leading cause of acquired blindness in working-age individuals. Recent work has revealed that neurodegeneration occurs earlier than vascular insult and that distal optic nerve damage precedes retinal degeneration and vascular insult. Since we have shown that optic nerve degeneration is reduced after optic nerve crush in Galectin-3 knockout (Gal-3 -/-) mice, we decided to investigate whether Gal-3 -/- could relieve inflammation and preserve both neurons and the structure of the retina and optic nerve following 8â¯weeks of diabetes. Diabetes was induced in 2-month-old male C57/bl6 WT or Gal-3 -/- mice by a single injection of streptozotocin (160â¯mg/kg). Histomorphometric retinal analyses showed no gross difference, except for a reduced number of retinal ganglion cells in WT diabetic mice, correlated to increased apoptosis. In the optic nerve, Gal-3 -/- mice showed reduced neuroinflammation, suggested by the smaller number of Iba1+ cells, particularly the amoeboid profiles in the distal end. Furthermore, iNOS staining was reduced in the optic nerves of Gal-3 -/- mice, as well as GFAP in the distal segment of the optic nerve. Finally, optic nerve histomorphometric analyses revealed that the number of myelinated fibers was higher in the Gal-3 -/- mice and myelin was more rectilinear compared to WT diabetic mice. Therefore, the present study provided evidence that Gal-3 is a central target that stimulates neuroinflammation and impairs neurological outcomes in visual complications of diabetes. Our findings provide support for the clinical use of Gal-3 inhibitors against diabetic visual complications in the near future.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Galectina 3/deficiencia , Inflamación/metabolismo , Neuroprotección/fisiología , Nervio Óptico/metabolismo , Retina/metabolismo , Animales , Apoptosis/fisiología , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Galectina 3/genética , Inflamación/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Nervio Óptico/patología , Retina/patologíaRESUMEN
A trauma to the mature central nervous system (CNS) often leads to persistent deficits, due to the inability of axons to regenerate after being injured. Increasing evidence suggests that pro-inflammatory and pro-apoptotic genes can present a major obstacle to promoting neuroprotection of retinal ganglion cells and consequently succeed in axonal regeneration. This study evaluated the effect of the absence of galectin-3 (Gal-3) on retinal ganglion cells (RGC) survival and axonal regeneration/degeneration after optic nerve crush injury. Two weeks after crush there was a 2.6 fold increase in the rate of cell survival in Gal-3-/- mice (1283±79.15) compared to WT animals (495.4±53.96). However, no regeneration was observed in the Gal-3-/- mice two weeks after lesion. Furthermore, axonal degeneration presented a particular pattern on those mice; Electron Microscopy (EM) analysis showed incomplete axon degeneration while the WT mice presented an advanced stage of degeneration. This suggests that the removal of the nerve fibers in the Gal 3-/- mice could be deficient and this would cause a delay in the process of Wallerian degeneration once there is a decrease in the number of macrophages/microglia in the nerve. This study demonstrates how the absence of Gal-3 can affect RGC survival and optic nerve regeneration/degeneration after lesion. Our results suggest that the absence of Gal-3 plays an important role in the survival of RGC and thus can be a potential target for therapeutic intervention in RGC neuroprotection.