Optimización de la transfección in vivo e in vitro mediante vectores no virales

En el mundo hay aproximadamente 285 millones de personas con discapacidad visual, lo que ha creado la necesidad de desarrollar nuevas terapias biotecnológicas para conseguir un tratamiento efectivo. La optogenética surge como una terapia viable para restaurar visión en retinopatías. Permite la activación inhibición del tejido nervioso mediante la expresión precisa de canales iónicos dependientes de luz en la membrana. Canales como la canalrodopsina-2 (ChR2) o su variante Catch, contienen una opsina que cuando es estimulada con una longitud de onda determinada (azul), provoca una modificación estructural que abre el canal permitiendo el flujo de los iones, resultando en una despolarización de la membrana y el disparo de un potencial de acción. En este estudio comprobamos la capacidad transfectiva tanto in vitro como in vivo usando magnetopartículas para introducir y expresar el plásm ido de la Catch

Deleterious Effect of NMDA Plus Kainate on the Inner Retinal Cells and Ganglion Cell Projection of the Mouse

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal “b” wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the “a” wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber.This research was funded by the Instituto de Salud Carlos III and co‐funded by the European Regional Development Fund (ERDF) within the “Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020” (grant numbers #RD16/0008/0016; #RD16/0008/0020; #FIS/PI 18‐00754)

Anti‐inflammatory action of dietary wild olive (Acebuche) oil in the retina of hypertensive mice

Inflammation plays a crucial role in the course of eye diseases, including many vascular retinopathies. Although olive oil is known to have beneficial effects against inflammatory processes, there is no information available on the anti‐inflammatory potential of the wild olive tree (namely, acebuche (ACE) for the primitive Spanish lineages). Here we investigate the anti‐inflammatory effects of ACE oil in the retina of a mouse model of arterial hypertension, which was experimentally induced by administration of L‐NAME (NG‐nitro‐L‐arginine‐methyl‐ester). The animals were fed supplements of ACE oil or extra virgin olive oil (EVOO, for comparative purposes). Retinal function was assessed by electroretinography (ERG), and different inflammation‐related parameters were measured in the retina and choroid. Besides significant prevention of retinal dysfunction shown in ERG recordings, ACE oil‐enriched diet upregulated the expression of the anti‐inflammatory mark-ers PPARγ, PPARα and IL‐10, while reducing that of major proinflammatory biomarkers, IL‐1β, IL‐ 6, TNF‐α and COX‐2. This is the first report to highlight the anti‐inflammatory properties of an ACE oil‐enriched diet against hypertension‐related retinal damage. Noteworthy, dietary supplementa-tion with ACE oil yielded better results compared to a reference EVOO.Ministerio de Ciencia e Innovación PID2019-109002RB-I00Junta de Andalucía 2020/275, 2021/188, CTS-584Universidad de Sevilla 2020/116

Adrenergic Modulation With Photochromic Ligands

© 2020 Wiley-VCH GmbH Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice

Programa de Doctorado en Ciencias de la Salud Universidad de Alcalá. Programa de Doctorado en Ciencias de la Visión

La creación y caracterización de nuevos modelos de experimentación animal es una necesidad acuciante hoy en día. Dichos modelos, pueden ser válidos como herramienta para el estudio de las enfermedades que afectan al ser humano, así como para la evaluación de nuevos agentes terapéuticos. En la presente Tesis Doctoral, intentaremos responder a la pregunta de si es posible la creación de modelos animales de neurodegeneración retiniana que asemejen enfermedades oculares que afectan al ser humano. Bien sea a través de modificaciones genéticas o bien induciendo lesiones que puedan evolucionar en una degeneración, en este trabajo, se caracterizarán nuevos modelos animales, comprobando su uso potencial como herramientas de investigación de patologías que cursan con neurodegeneración de las células de la retina. Así mismo, se procederá al estudio de nuevas moléculas con capacidades terapéuticas mediante el uso de estos modelos animales. Mediante el uso de diversas técnicas conductuales se pudo demostrar la afectación de diversas funciones visuales en los diversos modelos animales. Su capacidad para detectar espacios iluminados, la agudeza visual o incluso el reflejo pupilar evidenciaron el grado de disfunción debidas al curso de las diferentes lesiones neurodegenerativas. Las pruebas electrofisiológicas corroboraron los resultados obtenidos por las pruebas conductuales. Generalmente se observaron disminuciones en las amplitudes de las diversas ondas del Electrorretinograma de flash, así como disfunción en la capa de células ganglionares mediante el Electrorretinograma de pattern o incluso a nivel de corteza visual primaria, mediante el registro de potenciales evocados visuales. El marcaje inmunohistoquímico permitió mostrar daños celulares en las capas externas de la retina en el modelo mutante (Opn4-/- x Rd10) y tras la administración del agente de estrés oxidativo (yodato sódico), mientras que los daños del modelo inducido por inoculación de agentes excitotóxicos (NMDA/KA), mostro graves daños en las capas internas de la retina. Finalmente, dos nuevas moléculas con capacidades farmacológicas fueron estudiadas usando de base los modelos de degeneración de la retina externa Opn4-/- x Rd10 y yodato sódico), comprobando mediante varias pruebas conductuales y electrofisiológicas, su efecto funcional sobre estos animales. Los resultados obtenidos combinando toda la batería de pruebas realizada a cada modelo, tanto de naturaleza genética como de lesión inducida, validan los modelos animales descritos como herramientas prometedoras para el estudio de futuros agentes terapéuticos en enfermedades degenerativas de la retina

Phenotype Characterization of a Mice Genetic Model of Absolute Blindness

Recent technological development requires new approaches to address the problem of blindness. Such approaches need to be able to ensure that no cells with photosensitive capability remain in the retina. The presented model, Opn4−/− × Pde6brd10/rd10 (O×Rd) double mutant murine, is a combination of a mutation in the Pde6b gene (photoreceptor degeneration) together with a deletion of the Opn4 gene (responsible for the expression of melanopsin in the intrinsically photosensitive retinal ganglion cells). This model has been characterized and compared with those of WT mice and murine animal models displaying both mutations separately. A total loss of pupillary reflex was observed. Likewise, behavioral tests demonstrated loss of rejection to illuminated spaces and a complete decrease in visual acuity (optomotor test). Functional recordings showed an absolute disappearance of various wave components of the full-field and pattern electroretinogram (fERG, pERG). Likewise, visual evoked potential (VEP) could not be recorded. Immunohistochemical staining showed marked degeneration of the outer retinal layers and the absence of melanopsin staining. The combination of both mutations has generated an animal model that does not show any photosensitive element in its retina. This model is a potential tool for the study of new ophthalmological approaches such as optosensitive agents

Origin of Retinal Oscillatory Potentials in the Mouse, a Tool to Specifically Locate Retinal Damage

To determine the origin of oscillatory potentials (OPs), binocular electroretinogram (ERG) recordings were performed under light and dark adaptation on adult healthy C57BL/6J mice. In the experimental group, 1 μL of PBS was injected into the left eye, while the right eye was injected with 1 μL of PBS containing different agents: APB, GABA, Bicuculline, TPMPA, Glutamate, DNQX, Glycine, Strychnine, or HEPES. The OP response depends on the type of photoreceptors involved, showing their maximum response amplitude in the ERG induced by mixed rod/cone stimulation. The oscillatory components of the OPs were affected by the injected agents, with some drugs inducing the complete abolition of oscillations (APB, GABA, Glutamate, or DNQX), whereas other drugs merely reduced the oscillatory amplitudes (Bicuculline, Glycine, Strychnine, or HEPES) or did not even affect the oscillations (TPMPA). Assuming that rod bipolar cells (RBC) express metabotropic Glutamate receptors, GABAA, GABAC, and Glycine receptors and that they release glutamate mainly on Glycinergic AII amacrine cells and GABAergic A17 amacrine cells, which are differently affected by the mentioned drugs, we propose that RBC-AII/A17 reciprocal synapses are responsible for the OP generation in the ERG recordings in the mice. We conclude that the reciprocal synapses between RBC and AII/A17 are the basis of the ERG OP oscillations of the light response, and this fact must be taken into consideration in any ERG test that shows a decrease in the OPs’ amplitude

Origin of Retinal Oscillatory Potentials in the Mouse, a Tool to Specifically Locate Retinal Damage

To determine the origin of oscillatory potentials (OPs), binocular electroretinogram (ERG) recordings were performed under light and dark adaptation on adult healthy C57BL/6J mice. In the experimental group, 1 μL of PBS was injected into the left eye, while the right eye was injected with 1 μL of PBS containing different agents: APB, GABA, Bicuculline, TPMPA, Glutamate, DNQX, Glycine, Strychnine, or HEPES. The OP response depends on the type of photoreceptors involved, showing their maximum response amplitude in the ERG induced by mixed rod/cone stimulation. The oscillatory components of the OPs were affected by the injected agents, with some drugs inducing the complete abolition of oscillations (APB, GABA, Glutamate, or DNQX), whereas other drugs merely reduced the oscillatory amplitudes (Bicuculline, Glycine, Strychnine, or HEPES) or did not even affect the oscillations (TPMPA). Assuming that rod bipolar cells (RBC) express metabotropic Glutamate receptors, GABAA, GABAC, and Glycine receptors and that they release glutamate mainly on Glycinergic AII amacrine cells and GABAergic A17 amacrine cells, which are differently affected by the mentioned drugs, we propose that RBC-AII/A17 reciprocal synapses are responsible for the OP generation in the ERG recordings in the mice. We conclude that the reciprocal synapses between RBC and AII/A17 are the basis of the ERG OP oscillations of the light response, and this fact must be taken into consideration in any ERG test that shows a decrease in the OPs’ amplitude