Estudio de la respuesta glial y de las células ganglionares intrínsecamente fotosensibles en dos modelos animales de degeneración hereditaria de fotorreceptores y tras inyecciones intravítreas

Abstract

Purpose. To study the temporal course of photoreceptor cell death and macro and microglial reactivity in two rat models of retinal degeneration with different etiologies: the P23H- 1 and the Royal College of Surgeons (RCS) rat strains. To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in a rat model of inherited photoreceptor degeneration: theP23H-1 strain. To investigate the macro and microglial response of the normal rat retina after one or several intravitreal injections. Material y methods. To study the evolution of degeneration in two models of inherited retinal degeneration, we have used the P23H-1 and Royal College of Surgeon rat strains, and control age-matched animals: Sprague Dawley (SD) for the P23H1 rats and Pieval Virol Glaxo (PVG) for the RCS rats. The animals were sacrificed at different postnatal ages (P) (from P10 to P60), and their retinas were cryostat cross-sectioned. Sections were immunodetected with antibodies against: i) rhodopsin to label the rod outer segment, ii) L/M and S opsin to label the cone outer segments, iii) ionized calcium-binding adapter molecule1 (Iba1) to label microglial cells, iv) glial fibrillary acid protein (GFAP) to label macroglial cells, v) proliferating cell nuclear antigen (PCNA) to label cellular proliferation, and vi) isolectin B4 (IB4) to detect microglial cells and blood vessels. The numbers of photoreceptor nuclei rows in the outer nuclear layer and of microglial cells in the different retinal layers were quantified. To study the population of m+RGCs in P23H-1 rats we have used 30, 365, and 540 days old animals (P30, P365 and P540). As controls, we have used age-matched SD rats. The retinas were dissected as whole-mounts and immunodetected with antibodies against melanopsin and Brn3a to detect m+RGCs and the general population of RGCs, respectively. These populations were quantified and their distribution graphically represented with isodensity maps (for RGCs) and neighbour maps (for mRGCs). In addition, some morphometric dendritic parameters of m+RGCs were analysed. To investigate the response of macro and microglial cells after one or more intravitreal injections (IVI) we used SD rats. The left eye received one or three (one every 7 days) IVI of anti-rat VEGF (5 ?L; 0.015 ?g/?L), triamcinolone (2.5 or 5 ?L; 40 ?g/?L; Trigón¿ Depot), bevacizumab (5 ?L; 25 ?g/?L; Avastin¿), or their vehicles (PBS and balanced salt solution). Seven days after the last injection retinas were dissected as whole mounts and incubated with antibodies against: i) Iba1, ii) GFAP, and iii) vimentin (to label Müller cells). Macroglial cells were qualitatively analysed, while microglial cells were quantified using a semiautomatic method. In all studies retinas were examined with a fluorescence microscope, and some retinas that received IVI were observed with confocal microscopy. Results. In young animals with inherited retinal degeneration, photoreceptor degeneration starts earlier and progresses quicker in P23H-1 rats than in RCS rats. However, in both models, microglial cell activation occurs simultaneously with the initiation of photoreceptor death while GFAP over-expression in astrocytes and Müller cells begins later. As degeneration progresses, the total numbers of microglial cells in the retina increase and the numbers of microglial cells in the different layers increase in the outer retinal layers, but decrease in the inner retinal layers, more markedly in RCS rats. Microglial cells reach the outer nuclear and outer segment layers in both models. The higher number of microglial cells in dystrophic retinas cannot be fully accounted by intraretinal migration and PCNA immunodetection revealed microglial proliferation in both models, but more importantly in the RCS rats. Young (P30) P23H-1 rats had significantly lower numbers of Brn3a+RGCs than P30 SD control rats, while the population of m+RGCs was similar in both strains at this age. However, in adult P23H-1 rats there was a decrease in the number of m+RGCs and RGCs of 22.6% and 28.2% at 365 and 540 days of age, respectively. In addition, a decrease in morphometric dendritic parameters of m+RGCs was observed over time in both P23H-1 and P23H-3 rats (a rat line with a slower retinal degeneration). When analysing the co-expression of Brn3a and melanopsin in the P23H-1 rats, a significantly higher percentage of co-expression of both markers was found in m+RGCs already at P30 (3.31%) when compared to control animals (0.27%). This co-expression increased with age reaching 10.65% at P540. Finally, in the retinas treated with IVI we found that all the injected substances caused an important micro- and macroglial response locally at the injection site and all throughout the injected retina. This response was exacerbated by repeated IVI. In the contralateral non-injected eyes there was a microglial response as well, but it was milder than in the injected eye. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the treated retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-feet hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-feet hypertrophy was observed only in a specific area of the retina and was more extensive with triamcinolone or after repeated injections. Conclusions. In hereditary photoreceptor degenerations, the observed retinal changes vary depending on the etiopathogenic mechanism. In both models, photoreceptor death and microglial cell activation and migration occurred simultaneously, while the macroglial cell response is delayed. The activation of microglial cells in the degeneration process cannot be explained in the basis only of photoreceptor death: these cells participate more actively in the RCS model. Thus, this model is more inflammatory and would probably respond better to interventions aimed to inhibit microglial cells. Inherited photoreceptor degeneration was followed by secondary loss of RGCs labelled with Brn3a and mRGCs. Surviving mRGCs showed decreased dendritic morphometric parameters and increased coexpression of Brn3a and melanopsin. These phenotypic and molecular changes may represent an effort of mRGCs to resist degeneration and/or preferential survival of the cells capable of synthesizing Brn3a. Intravitreal injections cause micro- and macroglial responses that vary depending on the injected agent and the number of injections. The higher the number of injections, the greater the response. This inflammatory glial response may influence the effects of the injected substances on the retina.