Early Events in Retinal Degeneration Caused by Rhodopsin Mutation or Pigment Epithelium Malfunction: Differences and Similarities

To study the course of photoreceptor cell death and macro and microglial reactivity in two rat models of retinal degeneration with different etiologies. Retinas from P23H-1 (rhodopsin mutation) and Royal College of Surgeon (RCS, pigment epithelium malfunction) rats and age-matched control animals (Sprague-Dawley and Pievald Viro Glaxo, respectively) were cross-sectioned at different postnatal ages (from P10 to P60) and rhodopsin, L/M- and S-opsin, ionized calcium-binding adapter molecule 1 (Iba1), glial fibrillary acid protein (GFAP), and proliferating cell nuclear antigen (PCNA) proteins were immunodetected. Photoreceptor nuclei rows and microglial cells in the different retinal layers were quantified. Photoreceptor degeneration starts earlier and progresses quicker in P23H-1 than in RCS rats. In both models, microglial cell activation occurs simultaneously with the initiation of photoreceptor death while GFAP over-expression starts later. As degeneration progresses, the numbers of microglial cells increase in the retina, but decreasing in the inner retina and increasing in the outer retina, more markedly in RCS rats. Interestingly, and in contrast with healthy animals, microglial cells reach the outer nuclei and outer segment layers. 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 RCS rats. The etiology of retinal degeneration determines the initiation and pattern of photoreceptor cell death and simultaneously there is microglial activation and migration, while the macroglial response is delayed. The actions of microglial cells in the degeneration cannot be explained only in the basis of photoreceptor death because they participate more actively in the RCS model. Thus, the retinal degeneration caused by pigment epithelium malfunction is more inflammatory and would probably respond better to interventions by inhibiting microglial cells.Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15) and the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional “Una Manera de Hacer Europa” ISCIII-FEDER PI16/00380, PI16/00031, RD16/0008/0026, RD16/0008/0016, SAF2015-67643

Role of microglial cells in photoreceptor degeneration

Inherited photoreceptor degeneration in humans constitutes a major cause of irreversible blindness in the world. They comprise various diseases, but retinitis pigmentosa is the most frequently observed. Retinitis pigmentosa is commonly limited to the eye, where there is progressive photoreceptor degeneration, rods and secondarily cones. The mechanisms of cone and rod degeneration continue to be investigated, since most of the mutations causing retinitis pigmentosa affect rods and thus, the secondary death of cones is an intriguing question but, ultimately, the cause of blindness. Understanding the mechanisms of rod and cone degeneration could help us to develop therapies to stop or, at least, slow down the degeneration process. Secondary cone degeneration has been attributed to the trophic dependence between rods and cones, but microglial cell activation could also have a role. In this review, based on previous work carried out in our laboratory in early stages of photoreceptor degeneration in two animal models of retinitis pigmentosa, we show that microglial cell activation is observed prior to the the initiation of photoreceptor death. We also show that there is an increase of the retinal microglial cell densities and invasion of the outer retinal layers by microglial cells. The inhibition of the microglial cells improves photoreceptor survival and morphology, documenting a role for microglial cells in photoreceptor degeneration. Furthermore, these results indicate that the modulation of microglial cell reactivity can be used to prevent or diminish photoreceptor death in inherited photoreceptor degenerations

Estructura de la retina de atunes salvajes y nacidos en cautividad

Las especies de túnidos tienen una visión bien desarrollada. La morfología de la retina de juveniles de atún rojo (Thunnus thynnus) cultivados y salvajes fue investigada histológicamente. Los resultados obtenidos mostraron una mayor anchura del epitelio pigmentario y valores del índice de la retina más elevados en los individuos salvajes que en los individuos de cultivo. IntroducciónTuna species have well-developed vision. The morphology of the retina of Atlantic bluefin tuna juveniles (Thunnus thynnus) from cultured and wild fish were investigated histologically. The results showed a wider pigment epithelium and greater values of the retinal index in wild individuals than in cultured fish.FEM

Tracing the retina to analyze the integrity and phagocytic capacity of the retinal pigment epithelium

We have developed a new technique to study the integrity, morphology and functionality of the retinal neurons and the retinal pigment epithelium (RPE). Young and old control albino (Sprague-Dawley) and pigmented (Piebald Virol Glaxo) rats, and dystrophic albino (P23H-1) and pigmented (Royal College of Surgeons) rats received a single intravitreal injection of 3% Fluorogold (FG) and their retinas were analyzed from 5 minutes to 30 days later. Retinas were imaged in vivo with SD-OCT and ex vivo in flat-mounts and in cross-sections. Fifteen minutes and 24 hours after intravitreal administration of FG retinal neurons and the RPE, but no glial cells, were labeled with FG-filled vesicles. The tracer reached the RPE 15 minutes after FG administration, and this labeling remained up to 30 days. Tracing for 15 minutes or 24 hours did not cause oxidative stress. Intraretinal tracing delineated the pathological retinal remodelling occurring in the dystrophic strains. The RPE of the P23H-1 strain was highly altered in aged animals, while the RPE of the RCS strain, which is unable to phagocytose, did not accumulate the tracer even at young ages when the retinal neural circuit is still preserved. In both dystrophic strains, the RPE cells were pleomorphic and polymegathic.This study was supported by the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional “Una manera de hacer Europa” (PI16/00031, PI16/00380, PI19/00071, PI19/00203, SAF2015-67643-P, RD16/0008, RD16/0008/0026 and RD16/0008/0016) and by the Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15)

Different Ipsi-and Contralateral Glial Responses to Anti- VEGF and Triamcinolone Intravitreal Injections in Rats

Citation: Di Pierdomenico J, García-Ayuso D, Jiménez-López M, AgudoBarriuso M, Vidal-Sanz M, VillegasPérez MP. Different ipsi-and contralateral glial responses to anti-VEGF and triamcinolone intravitreal injections in rats. Invest Ophthalmol Vis Sci. 2016;57:3533-3544. DOI:10.1167/iovs.16-19618 PURPOSE. To investigate the glial response of the rat retina to single or repeated intravitreal injections (IVI). METHODS. Albino Sprague-Dawley rats received one or three (one every 7 days) IVI of anti-rat VEGF (5 lL; 0.015 lg/lL), triamcinolone (2.5 or 5 lL; 40 lg/lL; Trigón Depot), bevacizumab (5 lL; 25 lg/lL; Avastin), or their vehicles (PBS and balanced salt solution) and were processed 7 days after the last injection. Retinas were dissected as whole mounts and incubated with antibodies against: Iba1 (Ionized Calcium-Binding Adapter Molecule 1) to label retinal microglia, GFAP (Glial Fibrillary Acidic Protein) to label macroglial cells, and vimentin to label Müller cells. The retinas were examined with fluorescence and confocal microscopy, and the numbers of microglial cells in the inner retinal layers were quantified using a semiautomatic method. RESULTS. All the injected substances caused an important micro-and macroglial response locally at the injection site and all throughout the injected retina that was exacerbated by repeated injections. The microglial response was also observed but was milder in the contralateral noninjected eyes. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the ipsilateral retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-foot hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-foot hypertrophy was localized and more extensive with triamcinolone use or after repeated injections. CONCLUSIONS. Intravitreal injections cause micro-and macroglial responses that vary depending on the injected agent but increase with repeated injections. This inflammatory glial response may influence the effects of the injected substances on the retina

Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.This study was supported by the Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15), and the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional “una manera de hacer Europa” (SAF2015-67643-P, PI16/00380, RD16/0008/0026 and RD16/0008/0016)

Evaluation of the neuroprotective efficacy of the gramine derivative ITH12657 against NMDA-induced excitotoxicity in the rat retina

PurposeThe aim of this study was to investigate, the neuroprotective effects of a new Gramine derivative named: ITH12657, in a model of retinal excitotoxicity induced by intravitreal injection of NMDA.MethodsAdult Sprague Dawley rats received an intravitreal injection of 100 mM NMDA in their left eye and were treated daily with subcutaneous injections of ITH12657 or vehicle. The best dose–response, therapeutic window study, and optimal treatment duration of ITH12657 were studied. Based on the best survival of Brn3a + RGCs obtained from the above-mentioned studies, the protective effects of ITH12657 were studied in vivo (retinal thickness and full-field Electroretinography), and ex vivo by quantifying the surviving population of Brn3a + RGCs, αRGCs and their subtypes α-ONsRGCs, α-ONtRGCs, and α-OFFRGCs.ResultsAdministration of 10 mg/kg ITH12657, starting 12 h before NMDA injection and dispensed for 3 days, resulted in the best significant protection of Brn3a + RGCs against NMDA-induced excitotoxicity. In vivo, ITH12657-treated rats showed significant preservation of retinal thickness and functional protection against NMDA-induced retinal excitotoxicity. Ex vivo results showed that ITH12657 afforded a significant protection against NMDA-induced excitotoxicity for the populations of Brn3a + RGC, αRGC, and αONs-RGC, but not for the population of αOFF-RGC, while the population of α-ONtRGC was fully resistant to NMDA-induced excitotoxicity.ConclusionSubcutaneous administration of ITH12657 at 10 mg/kg, initiated 12 h before NMDA-induced retinal injury and continued for 3 days, resulted in the best protection of Brn3a + RGCs, αRGC, and αONs-RGC against excitotoxicity-induced RGC death. The population of αOFF-RGCs was extremely sensitive while α-ONtRGCs were fully resistant to NMDA-induced excitotoxicity

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

Objetivos Estudiar el curso temporal de muerte de los fotorreceptores y la respuesta de las células de macro y microglia en el inicio de la degeneración de la retina, en dos modelos animales de degeneración hereditaria de los fotorreceptores con diferentes mecanismos: la rata P23H-1 y la rata Royal College of Surgeons (RCS). Estudiar la población de células ganglionares de la retina intrínsecamente sensibles a la luz que expresan melanopsina (mCGRs) en uno de los modelos de degeneración hereditaria de fotorreceptores: la rata P23H-1. Investigar la respuesta de las células de macro y microglia de la retina de la rata adulta tras una o varias inyecciones intravítreas (IIV). Material y métodos. Para estudiar la evolución de la degeneración de la retina se utilizaron dos modelos de degeneración hereditaria de los fotorreceptores con paradigmas diferentes, la rata P23H-1 y la RCS, y como controles sanos la rata Sprague Dawley (SD) y la Pieval Virol Glaxo (PVG), respectivamente. Los animales fueron procesados entre los 10 y 60 días de edad y se realizaron secciones transversales en criostato de sus retinas. Las secciones fueron inmunodetectadas con anticuerpos contra; i) rodopsina para detectar los segmentos externos de los bastones, ii) Opsinas L/M y S para detectar los segmentos externos de los conos, iii) molécula ionizadora adaptadora de enlace de calcio1 (Iba1) para detectar las células de microglia, iv) proteína ácida fibrilar glial (GFAP) para detectar las células de macroglia , v) antígeno nuclear de células proliferantes (PCNA) para detectar células en división celular, vi) isolectina B4 (IB4) para detectar las células de microglia y vasos sanguíneos. Además, se cuantificaron las filas de núcleos de los fotorreceptores en la capa nuclear externa y las células de microglía en todas las capas de la retina. Para estudiar la población de mCGRs en las ratas P23H-1 y P23H-3 se utilizaron animales con 30, 365 y 540 días de edad y como control se utilizaron ratas SD con la misma edad. Tras diseccionar y montar a plano las retinas, éstas se inmunodetectaron con anticuerpos contra melanopsina y/o Brn3a para detectar la población de mCGR y la población general de células ganglionares de la retina (CGR), respectivamente. Finalmente, se cuantificaron dichas poblaciones y se representó gráficamente su distribución en la retina mediante el uso de mapas de isodensidad para las CGRs y mapas de vecinos para las mCGRs. Además, de estas últimas se analizó su arborización dendrítica. Para investigar la respuesta de las células de macro y microglia tras una o varias IIV se utilizaron ratas hembra adultas SD. Estas recibieron una o tres IIV (una cada 7 días) de anticuerpo anti VEGF derata (5 μl, 0,015 μg / μl), triamcinolona (2,5 o 5 μl, 40 μg / μL, Trigón Depot), bevacizumab (5 μL y 25 μg / µL, Avastin), o sus vehículos (PBS y solución salina equilibrada (BSS)). Las retinas se analizaron 7 días después de la última inyección, se montaron a plano y se incubaron con anticuerpos contra: i) Iba1, ii) GFAP y iii) vimentina (marcador específico de las células de Müller). Las células de macro y microglia fueron analizadas cualitativamente, además las células de microglia fueron analizadas cuantitativamente mediante un método semiautomático. En todos los estudios las retinas fueron examinadas con un microscopio de fluorescencia. En el estudio de las IIV también se utilizó además microscopía confocal. Resultados. Al analizar las retinas en los animales con degeneración retiniana, se observó que la degeneración de los fotorreceptores comenzaba antes y progresaba más rápidamente en las ratas P23H-1 que en las ratas RCS. Sin embargo, en ambos modelos de degeneración, la activación de las células de microglía ocurría simultáneamente a la muerte de los fotorreceptores; mientras que la sobreexpresión de GFAP en los astrocitos y células de Müller, comenzaba más tarde y con mayor intensida en estas últimas. A medida que progresaba la degeneración, en contraste con los animales sanos, encontramos células microgliales en las capas nuclear externa y de los segmentos externos de los fotorreceptores. Además, el número de células microgliales aumentó en la totalidad de la retina, pero disminuyendo en la retina interna y aumentando en la retina externa. Tanto el número total de células de microglia como la migración de las mismas desde las capas internas hacia las externas fue mayor en las ratas RCS. El mayor número de células microgliales en las retinas degeneradas no se puede explicar solamente con la migración intrarretiniana y además la inmunodeteccion de PCNA reveló proliferación microglial en ambos modelos, aunque más importantemente en las ratas RCS. Cuando se analizó la población de mRGCs y de CGRs en las ratas P23H-1 jovenes comparadas con los animales controles se observó una disminución significativa en las CGRs que expresan Brn3a, pero no de mRGCs. Sin embargo, en las ratas P23H-1 adultas se observó una disminución del número de mRGCs y de CGRs de un 22.6% y un 28,2% a los 365 y 540 días de edad, respectivamente. Además, con el tiempo se observó una disminución en los parámetros de arborización dendrítica de las mRGCs tanto en las ratas P23H-1 como en las ratas P23H-3 (cepa en la que la degeneración de la retina es más lenta). Al analizar la coexpresión de Brn3a y melanopsina en las ratas P23H-1 se encontró un porcentaje significativamente superior de coexpresión de ambos marcadores ya a 30 días de edad (3.31%) con respecto a los animales control (0.27%), además, este porcentaje de coexpresión aumentaba con la edad en las ratas P23H-1 (10,65% a 540 días de edad). Estos cambios celulares y de expresión se observaron solamente en los animales con degeneración hereditaria de los fotorreceptores (P23H-1), ya que en las ratas SD no se observó ningún cambio en la población general de CGR, ni en las población de mRGCs, ni en el porcentaje que mostró coexpresión (0.27%). Finalmente, en las retinas tratadas con las IIV se encontró en la zona de la inyección y a lo largo de toda la retina una importante respuesta de las células de macro y microglia, sin importar la sustancia inyectada; y esta respuesta fue mayor en las retinas que habían recibido varias inyecciones. También se observó una leve respuesta de las células de microglia tras las IIV en las retinas contalaterales que no habían sido inyectadas. Cuando se inyectó el anticuerpo humanizado bevacizumab, este causó una reacción/respuesta microglial tan fuerte que no se pudieron cuantificar las células de microglia. Al analizar la respuesta de las células de macroglia a las IIV se observaron dos tipos de respuesta: hipertrofia astrocítica e hipertrofia de los pies de las células de Müller. La hipertrofia de los astrocitos se observó en toda la superficie de las retinas inyectadas, mientras que la hipertrofia de los pies de las células de Müller sólo se observó en zonas bien definidas de la retina tras las inyecciones de triamcinolona y/o tras inyecciones repetidas. Conclusiones. En las degeneraciones hereditarias de los fotorreceptores la degeneración de la retina tiene un patrón diferente dependiendo del mecanismo etiopatogenico. En los dos modelos estudiados, la activación y migración de las células de microglia es simultánea a la muerte de los fotorreceptores, mientras que la respuesta de las células de macroglia es más tardía. La respuesta de la microglia no se puede explicar solamente en base a la muerte de los fotorreceptores ya que en los dos modelos existe una muerte severa de los fotorreceptores pero la respuesta de la microglia es mayor en las ratas RCS. Por lo tanto, en las ratas RCS la inflamación retininana es mayor y probablemente respondería mejor a un tratamiento antinflamatorio dirigido a la inhibición de las células de microglia. Tras la degeneración de los fotorreceptores hay una perdida secundaria de la población general de CGRs y de mCGRs. Las mCGRs supervivientes mostraron parámetros de arborización dendrítica disminuidos y aumento de la coexpresión de Brn3a y melanopsina. Estos cambios fenotípicos y moleculares pueden representar un esfuerzo de las mCGRs capaces de expresar Brn3a para resistir a la degeneración y / o supervivencia preferencial de las mCGRs. Las inyecciones intravítreas causan una respuesta en las células de macro y microglia que varía dependiendo de las sustancias inyectadas y del número de inyecciones. A mayor número de inyecciones mayor respuesta. Además la respuesta inflamatoria de la glía puede influir en los efectos de las sustancias inyectadas en la retina. SUMMARY. 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 μL; 0.015 μg/μL), triamcinolone (2.5 or 5 μL; 40 μg/μL; Trigón® Depot), bevacizumab (5 μL; 25 μg/μL; 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

Retinal Ganglion Cell Death as a Late Remodeling Effect of Photoreceptor Degeneration

Inherited or acquired photoreceptor degenerations, one of the leading causes of irreversible blindness in the world, are a group of retinal disorders that initially affect rods and cones, situated in the outer retina. For many years it was assumed that these diseases did not spread to the inner retina. However, it is now known that photoreceptor loss leads to an unavoidable chain of events that cause neurovascular changes in the retina including migration of retinal pigment epithelium cells, formation of “subretinal vascular complexes”, vessel displacement, retinal ganglion cell (RGC) axonal strangulation by retinal vessels, axonal transport alteration and, ultimately, RGC death. These events are common to all photoreceptor degenerations regardless of the initial trigger and thus threaten the outcome of photoreceptor substitution as a therapeutic approach, because with a degenerating inner retina, the photoreceptor signal will not reach the brain. In conclusion, therapies should be applied early in the course of photoreceptor degeneration, before the remodeling process reaches the inner retina

Glial Cell Activation and Oxidative Stress in Retinal Degeneration Induced by β-Alanine Caused Taurine Depletion and Light Exposure

International audienceWe investigate glial cell activation and oxidative stress induced by taurine deficiency secondary to β-alanine administration and light exposure. Two months old Sprague-Dawley rats were divided into a control group and three experimental groups that were treated with 3% β-alanine in drinking water (taurine depleted) for two months, light exposed or both. Retinal and external thickness were measured in vivo at baseline and pre-processing with Spectral-Domain Optical Coherence Tomography (SD-OCT). Retinal cryostat cross sections were immunodetected with antibodies against various antigens to investigate microglial and macroglial cell reaction, photoreceptor outer segments, synaptic connections and oxidative stress. Taurine depletion caused a decrease in retinal thickness, shortening of photoreceptor outer segments, microglial cell activation, oxidative stress in the outer and inner nuclear layers and the ganglion cell layer and synaptic loss. These events were also observed in light exposed animals, which in addition showed photoreceptor death and macroglial cell reactivity. Light exposure under taurine depletion further increased glial cell reaction and oxidative stress. Finally, the retinal pigment epithelial cells were Fluorogold labeled and whole mounted, and we document that taurine depletion impairs their phagocytic capacity. We conclude that taurine depletion causes cell damage to various retinal layers including retinal pigment epithelial cells, photoreceptors and retinal ganglion cells, and increases the susceptibility of the photoreceptor outer segments to light damage. Thus, beta-alanine supplements should be used with caution