Respuesta de las neuronas retinianas a la lesión y su neuroproteccióncaracterización de las células ganglionares alfa de la retina

Résumé

Introduction: Injury to the central nervous system (CNS) of the adult mammal cause, in most instances, unrepairable and permanent damage. The visual system and, in particular, the retina is often used as suited model to study the neurodegenerative processes underlying CNS pathophysiology, but the necessary tools are not always available. Objectives: Here we propose a new tool to study intracellular calcium flux in retinal bipolar cells (BCs). We also characterize alpha retinal ganglion cells (αRGC) in the pigmented mouse retina and study the survival of these cells in a model of intraorbital complete optic nerve transection (ONT) and excitotoxicity induced by intravitreal administration of N-methyl-D-aspartic acid (NMDA). In these models, we evaluated the neuroprotective effects of the systemic administration of the molecules 7,8-Dihydroxyflavone (DHF) which is a potent agonist of the TrkB receptor, ITH12657 which is a mild calcium voltage channel blocker, and ITH15004 which is a mild and selective P2X7 receptor antagonist. In addition, we characterize a model of acute ocular hypertension (AOH) in the adult albino rat and pigmented mouse. We analyze the cell death pathways involved following AOH and the protective effects of the molecules DHF, ITH12657 and ITH15004. Materials and methods: To study Ca2+ influx we injected AAV2.Ple265.GCaMP6f.WPRE virus and analyzed the changes that occurred in the axonal terminals of bipolar cells (BC) after illumination in vivo with 2-photon microscopy. To study the effects on the retina of ONT, NMDA or AOH and its protection we used in vivo techniques such as full-field electroretinogram (ERG) and spectral-domain optical coherence tomography (SD-OCT) and ex vivo techniques such as immunohistochemistry and quantitative polymerase chain reaction (qPCR). Results: i) AAV2.Ple265. GCaMP6f is a good tool to study calcium flux in CB terminals, and it was possible to observe that microdomains of the same terminal do not respond equally to the same light stimulus; ii) αRGC represent 5.23% of the pigmented mouse population of RGCs, those that respond to light in a sustained manner are more frequent in the superior temporal quadrant while those that respond transiently populate mainly the mid-peripheral part of the retina; iii) OHT causes permanent and severe damage early in the inner retina and later in the outer retina. It courses with 6 types of cell death and the combination of DHF, ITH12657 and ITH15004 prevents retinal degeneration; iv) NMDA excitotoxicity causes unequal degeneration in αRGC subtypes. While the αRGC-OFF almost disappear after NMDA excitotoxicity, αRGC-ONt are not affected, and the population of αRGC-ONs is depleted by 73%. ITH12657 rescues αRGC-ONs up to 21 days and DHF up to 7 days; v) ONT results in an almost disappearance of the αRGC-OFF population and in the loss of 75% of the αRGC-ONs. DHF rescues 66% of the αRGC-ONs up to 14 days and improves RGC functionality up to 21 days. Conclusions: Visual information that is perceived by a single cone bipolar cell is sorted at its axonal terminals. αRGCs represent 5.2% of the total pigmented mice RGC population, and present different resilience depending on the subtype and the kind of injury. The molecules, DHF, ITH12657 and ITH15004 exhibit beneficial retinal effects in the retinal injury models of ONT, OHT or NMDA and could potentially be used in the future to treat glaucoma.