Curso de activación glial y muerte de las células ganglionares de la retina en dos modelos de degeneración

Resumen

INTRODUCTION - Optic nerve crush (ONC) is a model of degeneration of retinal ganglion cells (RGCs). As a consequence of RGCs death, macroglial and microglial cells (MCs) become activated and change their morphology, and the latter migrate and phagocytose the dead RGCs. The response to ONC is not limited to the injured retina. The contralateral retina responds like the injured retina, but in a modest way. Thus, after ONC in the contralateral retina, activation of MCs, appearance of phagocytic microglia, activation of astrocytes and Müller cells and loss of RGCs are observed. Organotypic retinal cultures (ROCs) are being used as an in vivo surrogate to study the loss of RGCs. OBJECTIVES - 1) To study the glial response in axotomised retinas and retinas contralateral. 2) To study and compare the course of RGC death and glial response in ROCs and axotomised retinas. 3) To analyse and quantitatively compare the morphology of MCs in intact, axotomised, contralateral and in ROCs. MATERIAL AND METHODS - Adult pigmented C57Bl/6 mice were used. ONC was performed at 0.5 mm and analysis times were 1 to 45 days in vivo and 1 to 7 days in vitro. Immunofluorescence was performed on cut retinas, flat retinas and retinal explants with different markers for the study of different cell populations such as MCs, astrocytes and Müller cells and RGCs. The quantification of RGCs was performed automatically and the quantification of microglia was performed manually. The morphology of the MCs and the fluorescence intensity were analysed using Image J software. RESULTS - In the course of glial activation after ONC, the two states of the MCs were characterised. In lesioned retinas, the density of MCs-Iba1+ and MCs-CD68+ in the GCL increased significantly and returned to normal at 45 days. In contralateral retinas, the density of Iba1+-MCs increased significantly at 5 days, while the density of MCs-CD68+ was significantly higher after injury. In cross-sections, the density of MCs increased significantly in lesioned retinas, but not in contralateral retinas. Astrocytes and Müller cells were hypertrophied in both retinas. Comparative analysis of the course of RGC death and microglial activation between ROCs and axotomised retinas showed a similar loss of RGCs-Brn3a+ in both models. In vivo, Brn3a and RBPMS expression was parallel, however, after 5 days the in vitro RBPMS signal is maintained. When testing other RGC markers, it was observed that the number of immunodetected RGCs was the same as when using RBPMS. Quantification of MCs in ROCs showed a lower density than after ONC. Macroglial activation in ROCs occurs earlier than in vivo, and encompasses the entire retina, whereas after ONC it is confined to the RGC complex. Comparative analysis of microglial morphology between the two models showed qualitative changes in the morphology of MCs-Iba1+. In all three experimental groups, (lesioned, contralateral and ROCs retinas), all analysed parameters of the CMs differed from resting MCs. In each experimental group, the activation of the MCs followed specific kinetics, and the morphological changes in the contralateral retinas were more subdued than in the axotomised retina, and in the axotomised retina than in the ROCs. CONCLUSIONS - ONC triggers a bilateral and persistent activation of RGCs-Iba1+ and an opposite response of M2 RGCs between both retinas. The death of RGCs in both models is similar for up to 5 days, which means that they may be useful for studying new RGC-protective drugs. In all experimental groups, the MCs undergo changes in the size, number and length of their extensions. These morphological alterations are most marked in ROCs followed by axotomised retinas and finally those found in contralateral retinas.