Estudio de la degeneración de las células ganglionares de la retina tras axotomía del nervio ópticoensayo de nuevas terapias celulares y farmacológicas

Abstract

The retina is an extension of the Central Nervous System (CNS) and is easily accessible which makes it a unique model of the general Nervous System. The retinal ganglion cells (RGCs) form the optic nerve (ON) and send visual information to the brain. CNS neurons are unable to divide, so there is no possibility to form new neurons and replacing those that are damaged. The ON lesion causes the degeneration of a large part of the RGC population in two phases, a quick in the first 14 days and a slow one, and that there is a response in the contralateral eye. In rats, it has been documented that the time course of death in RGCs varies depending on the distance at which ON is sectioned from the eyeball. Microglia cells play an essential role in the innate immune response and neuroinflammation in the CNS. Neurotrophic factors are endogenous growth factors necessary for the survival of neurons. Retinoids represent a family of vitamin A-derived compounds, which have been shown to play a role in both proliferation and differentiation in adult neurogenesis. Minocycline is a tetracycline antibiotic that relieves inflammatory response and provides neuroprotection. Stem cell therapy is an ideal tool for CNS repair, as stem cells are capable of self-regenerating and differentiating into other cell types, as well as secreting various factors that promote neuron survival. OBJECTIVES: The general objectives of this thesis are: i/ to characterize organotypic retinal cultures as a model for the study of the RGC population; ii/ to analyze the behavior of the RGC death curve after injury at two different distances, both in the injured eye and in the contralateral eye; iii/ to study the survival of stromal cells derived from adipose tissue after intravitreal injection, and their role in the course of RGC death after axotomy; iv/ to study the behaviour of ciliary body cells (CB), their neurospheres (NS-CB), the neurospheres of subventricular zone cells (NS-SVZ) and ESR1 embryonic cells, after injection into the vitreous in axotomized and depleted retinas v/ to analyse the role of NLRP3 inflammatory proteins in RGCs; vi/ to analyze the role of TNFR1 antagonism in the survival of RGCs in axotomized retinas; vii/ preliminary neuroprotective studies of retinoic acid receptor agonism and the long-term study of BDNF. MATERIALS AND METHODS: For the accomplishment of the experiments were used: male pigmented mice (C57BL/6) with a weight between 25-35g, male pigmented transgenic mice with expression of the green fluorescent protein under the promoter of the actin, KO mice of the NLRP3 protein, KO mice of the ASC protein and KO mice of the Caspase 1/11 protein, with ages between 8 and 12 weeks of age. The number of retinas analyzed by group/stretch/time interval varies between the different studies from 4 to 20. A model of axonal damage of the RGCs was performed: by ON crush (ONC). ONC was performed 0.5 mm or 2 mm from the eyeball. Analysis times after axotomy ranged from 3 days to 4 months. The drugs or cells were administered by intravitreal or intraperitoneal injection. The anatomical analysis of the retinas was carried out mainly on complete retinas dissected at the immunodetected plane with antibodies anti-Brn3a or Rbpms (RGCs), anti-Iba-1 (microglia), anti-GFAP (astrocytes), anti-NeuN (neurons) and anti-GFP. Some retinas were dissected fresh to analyze the mRNA level by quantitative polymerase chain reaction or to perform organotypic retinal cultures (ORC). The samples were photographed with an epifluorescence microscope and a confocal microscope. The retinal photomontages were reconstructed from 154 single images (11x14) captured with the 20x lens. Brn3a+-RGCs were quantified and topographic maps were studied using automatic routines. RESULTS AND CONCLUSIONS. Objective 1: In the ORCs there is a loss in the first 24 hours of approximately 60% of the RGCs, followed by a gradual phase of loss up to 21 days of cultivation. This course resembles the pattern of death observed in vivo, but 2-3 times faster. The retina in ORCs suffers a degeneration in all its layers as more time passes in the culture. Objective 2: Although the course of RGC loss in injured retinas does not differ according to the distance at which ONC is performed, in contralateral retinas there is a significant loss of 15% that occurs earlier when axotomy is performed closer to the contralateral eye. Systemic treatment with minocycline or meloxicam rescues RGCs in contralateral retinas but not in injured retinas. Objective 3: Mesenchymal stromal cells derived from adipose tissue (Ad-MEC) are observed in vivo in the vitreous by OCT up to 21 days after intravitreal transplantation. Ex vivo, Ad-MEC are found forming a membrane over the retina, both in intact and injured retinas. Intravitreal injection of Ad-MEC did not produce toxicity to the RGCs, at least until 21 days after transplantation. RGC death course after ONC did not differ between the control group and the treated group. Objective 4: Both CB, NS-CB and NS-SVZ cells survive in the retina forming an epirretinal membrane without producing toxicity in the RGC population and causing an increase in survival after ON axotomy, with special attention to NS-CB which achieve a high percentage of survival. CB and NS-CB cells penetrate the ganglionic layer of depleted retinal cells and are able to differentiate into Brn3a+ and Neun+ cells, while NS-SVZ have less differentiating capacity but are able to penetrate up to the internal nuclear layer. Objective 5: The deficit of the NLRP3 inflammatory proteins does not cause any change in the total number of quantified RGCs compared to wild retinas. In the retinas ASC-/- and NLRP3-/- no difference is observed in the course of RGC death after ONC compared to the wild type retinas. The Casp1/11-/- group has a survival of up to 50% of RGCs compared to wild retinas at 9 and 21 days after injury. Objective 6: After ONC, there is an increase in the gene expression of TNFR1 and TNFalpha. Both systemic and direct treatment with the TNFR1 antagonist (R7050) increases RGC survival after axotomy. BDNF and double treatment (R7050 and BDNF) produce a great neuroprotective effect in RGC after axotomy, both at 5 and 14 days after ONC. Objective 7: Treatment with agonists from RARalpha and RARbeta promotes the survival of RGCs after ONC, with daily intravitreal or intraperitoneal injections. An intravitreal injection of BDNF after ONC neuroprotects part of the RGCs up to 45 days post-injury.