DigitalUM :: Browsing by Subject "Retina"

Browsing by Subject "Retina"

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A computerized analysis of the entire retinal ganglion cell population and its spatial distribution in adult rats
(Elsevier, 2009-01) Mayor Torroglosa, S.; Jiménez López, M.; Avilés Trigueros, Marcelino; Holmes, T. M.; Lund, R. D.; Villegas Pérez, M. P.; Vidal Sanz, M.; Salinas Navarro, Manuel Ángel; Anatomía Humana y Psicobiología
In adult albino (SD) and pigmented (PVG) rats the entire population of retinal ganglion cells (RGCs) was quantified and their spatial distribution analyzed using a computerized technique. RGCs were back-labelled from the optic nerves (ON) or the superior colliculi (SCi) with Fluorogold (FG). Numbers of RGCs labelled from the ON [SD: 82,818 ± 3,949, n = 27; PVG: 89,241 ± 3,576, n = 6) were comparable to those labelled from the SCi [SD: 81,486 ± 4,340, n = 37; PVG: 87,229 ± 3,199; n = 59]. Detailed methodology to provide cell density information at small scales demonstrated the presence of a horizontal region in the dorsal retina with highest densities, resembling a visual streak.
Open Access
A high-density narrow-field inhibitory retinal interneuron with direct coupling to Müller glia
(Society for Neuroscience, 2021-07-14) Grimes, William N. ; Aytürk, Didem Göz ; Hoon, Mrinalini ; Yoshimatsu, Takeshi ; Gamlin, Clare ; Carrera, Daniel ; Nath, Amurta ; Ahlquist, Richard M. ; Sabnis, Adit ; Berson, David M. ; Diamond, Jeffrey S. ; Wong, Rachel O. ; Cepko, Connie ; Rieke, Fred ; Nadal-Nicolás, Francisco Manuel; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica; Facultad de Medicina
Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features, such as edges or directed motion, by mediating excitatory and inhibitory interactions between input (i.e., bipolar) and output (i.e., ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation, and neurovascular control. Here, we report that, in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labeling of nearby Müller glia, leading us to suggest the name "Müller glia-coupled amacrine cell," or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus showed selective connections between MACs and a subpopulation of retinal ganglion cell types. Visually evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function. SIGNIFICANCE STATEMENT Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics, and serial electron microscopy provide new information about the neuron's synaptic partners and physiological responses.
Open Access
Adaptive changes in the visual cortex after photoreceptor degeneration in retinitis pigmentosa
(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Martinez Galan, Juan R.; Caminos, Elena; Biología Celular e Histología
Retinitis pigmentosa (RP) is a group of hereditary disorders that cause progressive retinal degeneration, affecting the rods and, subsequently, the cones, which results in progressive vision loss. RP is genetically heterogeneous and is inherited in an autosomal dominant, autosomal recessive, X-linked, or sporadic non-Mendelian manner. The recent advance-ments in repairing damaged retinas highlight the necessity of understanding the impact of photoreceptor degeneration on the visual cortex. This is because functional vision may not be adequately restored if this region is significantly impaired prior to treatment. In the present review, we have analyzed the rodent models of RP that have been most frequently used and the physiological and morphological changes occurring in both humans and rodents with this disorder. Following visually evoked stimulation, the processing of visual information in the primary visual cortex (V1) of individuals with RP is altered due to modifications in the transduction of the signal originating in the degenerated retina. Moreover, alterations in the intrinsic electro-physiological properties of cortical neurons and neural circuits have also been documented. Finally, several neurochemical and/or morphological changes are observed in synaptic structures associated with pyramidal neurons and in select inhibitory interneurons. Nevertheless, despite the physiological and morphologi-cal changes that have been described, the impact of RP on the visual cortex does not inevitably result in irreversible damage, as the alterations do not appear to be particularly severe. Brain plasticity is more restricted in adults; however, remodeling of the visual cortex in mice and humans is possible, which encourages further work on therapies capable of partially restoring the lost visual function.
Open Access
Análisis de la degeneración de los fotorreceptoes en modelos experimentales de retinosis pigmentaria, degeneración macular asociada a la edad y glaucoma =Analysis of photoreceptor degeneration in experimental models of retinitis pigmentosa, aging macular degeneration and glaucoma
(2015-07-24) Ortín Martínez, Arturo; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Villegas Pérez, María Paz; Facultad de Medicina
. Objetivo. Determinar el número total y la topografía de la población de conos en roedores adultos utilizando rutinas automatizadas que nos permita investigar objetivamente los efectos de diferentes modelos experimentales de patologías humanas tales como la degeneración macular asociada a la edad (DMAE), retinosis pigmentaria (RP) y la neuropatía óptica glaucomatosa (NOG) sobre la población de fotorreceptores. Material y métodos. Se han utilizado cinco cepas distinta de roedores: Sprague-Dawley (SD), Piebald Virol Glaxo (PVG) y la rata transgénica P23H-1, y ratones Swiss y C57/BL6. Todos los experimentos y procedimientos se han realizado bajo el estricto cumplimiento de las recomendaciones de la “Guide for the Care and Use of Laboratory Animals of the Association for Research in Vision and Ophthalmology (ARVO)” y la “European Union guidelines for the use of animals in research”, y todos los procedimientos utilizados han sido previamente aprobados por el Comité Ético de Experimentación Animal (CEEA) de la Universidad de Murcia. Se han utilizado diferentes técnicas para el estudio histológico y morfológico y se han analizado en retinas montadas a plano y en secciones transversales; se han utilizado tecnologías de imagen avanzadas como la Spectral Domain Optical Coherence Tomography (SD-OCT)y se ha realizado análisis de proteínas mediante Western Blot. Como modelo de RP se ha utilizado la rata transgénica P23H-1, esta rata es portadora de una mutación autosómica dominante en el gen de la rodopsina que causa distrofia y muerte de los fotorreceptores. Se ha desarrollado un nuevo modelo experimental útil para la comprensión de la patología de la DMAE, la fototoxicidad localizada de los fotorreceptores tipo cono inducida por LED (FTIL). Por último se ha utilizado un modelo experimental de hipertensión ocular (HTO) inducida mediante láser que permite la evaluación de los efectos de la NOG sobre la población de fotorreceptores. Resultados. El número medio de conos que expresan la opsina-L es de 231.736 ± 14.517 en la rata SD; 239.939 ± 6.494 en la rata PVG; 117.424 ± 17.721 en el ratón Swiss y 135.155 ± 8.742 en el ratón C57/BL6. El número medio de conos que expresan la opsina-S es de 41.028 ± 5.074 en la rata SD; 27.316 ± 2.235 en la rata PVG; 146.682 ± 24.958 en ratones Swiss y 119.616 ± 8.756 en ratones C57/BL6. El porcentaje de conos duales en la rata SD es del 3,2%, del 2,9% en la PVG, de un 73% en el ratón Swiss y de un 40% en el C57/BL6. En todas las cepas de ambas especies existe un paralelismo en la distribución de las células ganglionares de retina (CGR) y los conos-L. La topografía de los conos-L en todas las cepas de rata y ratón analizadas es similar, se observan zonas de alta densidad en el eje nasotemporal superior, las densidades medias alrededor del nervio óptico y un descenso de densidad gradual desde las zonas centrales hacia las periféricas. Sin embargo, existen claras diferencias en la distribución de los conos-S entre las especies y cepas analizadas. El la rata P23H-1, la degeneración de los bastones ocurre antes que la de los conos y de forma rápida: primero con el acortamiento de los segmentos externos, a P30 existe una gran pérdida de bastones y a P180 la pérdida es prácticamente en la totalidad de la retina exceptuando la extrema periferia. La degeneración de bastones y conos está espaciotemporalmente relacionada, ocurre en forma de anillos que aparecen alrededor de P90 y se extiende por toda la retina. A P180, los anillos de degeneración son más abundantes en la retina ecuatorial y de mayor tamaño en la retina dorsal. En un nuevo modelo in vivo de fototoxicidad focal de los fotorreceptores inducido por LED, la SD-OCT muestra un daño en una región circular situado en la retina superotemporal. En esta región se observa una disminución progresiva del espesor de la retina desde 183,4 ± 5 mm (12 h) hasta 114,6 ± 6 mm (7 d). Las secciones transversales muestras una pérdida masiva de bastones y conos en la región dañada por la luz. En las retinas montadas a plano se observa una región circular con disminución del número de conos-L y conos-S. En este área circular en las retinas izquierdas y en la región correspondiente de la retina control derecha, el número total de conos-L o conos-S es de 7.118 ± 842 ó 661 ± 125 en las retinas fotoexpuestas (n=7) y de 14.040 ± 1.860 ó 2.255 ± 193 en las retinas control (n=7), respectivamente. Aunque el CNTF no, la brimonidina, el BDNF, PEDF y el bFGF muestran efectos neuroprotectores significativos sobre los conos-L y conos-S. La HTO provoca sectores con su vértice en el disco óptico carentes de CGR Brn3a pero que aún contienen gran número de núcleos DAPI positivos. Los niveles de todas las opsinas disminuyen a las 2 semanas y esta disminución progresa hasta el 20% de los niveles basales a los 3 meses. Las secciones transversales revelan áreas focales de degeneración en las capas externas de la retina (CER). Las CGR supervivientes a los 15 días representan aproximadamente el 28% y no cambian con el tiempo, mientras que de las poblaciones de conos-L y conos-S sobreviven un 80% y un 65% a un mes o un 35% y un 20% a 6 meses, respectivamente. Conclusiones. Se ha establecido, por primera vez, el número total y la distribución topográfica de los conos-L y conos-S en dos cepas de rata y dos de ratón y se ha demostrado el paralelismo topológico de la distribución de los conos-L y las CGR. Se han proporcionado las bases para estudiar la degeneración de conos y su prevención en condiciones patológicas. Se ha descrito por primera vez que, en la rata P23H-1, la degeneración de bastones y conos está espaciotemporalmente relacionada y se produce en anillos. La pérdida de conos sigue a la pérdida de bastones, que comienza de forma temprana, incluso antes de P30, la primera edad analizada. Las características de los anillos sugieren que la degeneración secundaria de conos está influenciada por la localización en la retina y / u otros factores intrínsecos o extrínsecos. Se ha evidenciado que la FTIL provoca una pérdida de conos y bastones y es un modelo fiable, cuantificable y reproducible para estudiar la degeneración de los fotorreceptores. La administración intravítrea de BDNF, PEDF o bFGF, o la administración tópica de brimonidina proporcionan neuroprotección significativa sobre los conos, en este modelo. Se ha demostrado que la HTO induce una pérdida selectiva de CGR en la capa de la CGR que no progresa después de 1 mes, mientras que los conos-L y conos-S presentan una pérdida progresiva hasta los 6 meses. Por lo tanto, HTO provoca graves daños tanto en las capas más internas como en las CER. SUMMARY. Purpose. To determine the total number and topography of the cone population in two rat and two mouse strains using automated routines which allowed us to investigate objectively the effects of different experimental models of human pathologies such as Aging Macular Degeneration (AMD), Retinitis Pigmentosa (RP) and Glaucomatous Optic Neuropathy (GON) on the photoreceptor population. Material y methods. A total of 303 rats and 23 mice were used in this thesis, five different strains of rodents were used: albino Sprague-Dawley (SD), pigmented Piebald Virol Glaxo (PVG) and P23H-1 transgenic rats, albino Swiss and pigmented C57/BL6 mice. All experimets and procedures were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Association for Research in Vision and Ophthalmology (ARVO) and the European Union guidelines for the use of animals in research, and all the protocols were approved by the Ethical and Animal Studies Committee of the University of Murcia. Several techniques such as, the identification of the three different types of photoreceptors and other retinal populations by immunohistofluorescence analyzed in whole flat-mounted retinas and in oriented radial sections, classical staining as H-E, imaging advanced technologies as Spectral Domain Optical Coherence Tomography (SD-OCT), and protein analysis by western blot, have been used. In this thesis the P23H-1transgenic rat has been used as a model of RP, this animal bears an autoasomal dominat mutation in the rhodopsin gene (proline to histidine substitution at codon 23 of the rodopsin protein) that causes photorecptor dystrophy and death. A new experimental model useful to understand the AMD pathology, has been developed for this thesis, Light Emmitting Dioede (LED)-induced cone-photoreceptor phototoxicity (LIP), the blue-light LED exposition on the rat retina causes a damage-area located in the retinal zone with maximun L-cones densities and a cone to rod ratio similar to the human macular fovea. And finally, an experimental model of Laser-induced ocular hypertension developed recently in our Laboratory of Experimental Ophthalmology at the University of Murcia has been used to understand the effects of GON on the cone population. Results. The mean number of L-opsin+cones is 231,736 ± 14,517 in SD rat; 239,939 ± 6,494 in PVG rat; 117,424 ± 17,721 in Swiss mouse and 135,155 ± 8,742 in C57/BL6 mouse. The mean number of S-opsin+cones 41,028 ± 5,074 in SD rat; 27,316 ± 2,235 in PVG rat; 146,682 ± 24,958 in Swiss mouse and 119,616 ± 8,756 in C57/BL6 mouse. The percentage of dual cones is 3.2% in SD rat; 2.9% in PVG rat; 73% in Swiss mouse and 40% in C57/BL6 mouse. In all strains, and both species, there is a parallel distribution of retinal ganglion cells (RGC) and L-cones. The topography of L-cones is similar in all strains of rats and mice analyzed, the highest densities are observed in the superior nasotemporal axis, medium densities around the optic nerve, and this density gradually decreases from the center to the periphery. However, obvious differences are found in S-cones distribution. While in the two rat strains there is a increasing gradient of S-cones density along the inferonasal quadrant and the highest densities are found in the retinal rim, in the Swiss mouse strains S-cones are abundant in the dorsal retina although their highest densities are ventral but the C57/BL6 mouse shows a low number of S-cones in the dorsal retina and very dense population in the ventral retina, being densest in its nasal aspect. In P23H-1 rats, rod degeneration occurs rapidly: first the rod outer segment shortens, at P30 there is extensive rod loss, and by P180 rod loss is almost complete except for the most peripheral retina. The numbers of L cones are, at all postnatal ages, lower in P23H-1 rats than in control SD rats, and decrease significantly with age (by P180). Rod and cone degeneration is spatiotemporally related and occurs in rings that appear already at P90 and spread throughout the entire retina. At P180, the rings of rod-cone degeneration are more abundant in the equatorial retina and are larger in the dorsal retina. In a novel in vivo model of focal LED-induced photoreceptor phototoxicity SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4 ± 84.5 mm (at 24 hours) to 1,407.7 ± 52.8 mm (at 7 days). This region had a progressive thickness disminution from 183.4 ± 5 mm (at 12 h) to 114.6 ± 6 mm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118 ± 842 or 661 ± 125 for the LED exposed retinas (n=7) and 14,040 ± 1,860 or 2,255 ± 193 for the fellow retinas (n=7), respectively. Brimonidine, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- and S-cones. Ocular hypertension (OHT) resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3aRGC but with large numbers of DAPI+nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of outer retinal layers (ORL) degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the L-cone and S- populations diminished to 80% and 65%, or to 35% and 20% at 1 or 6 months, respectively. Conclusions. It has been established, for the first time, the total number and the topographical distribution of S- and L-cones in two rat and two mouse strains and demonstrated the correlation of L-cones and RGC spatial distribution. It has been provided the basis to study cone degeneration and its prevention in pathologic conditions. It has been described for the first time that in the P23H-1 rat, rod and cone degeneration is spatiotemporally related and occurs in rings. Cone loss follows rod loss and starts very soon, even before P30, the first age analyzed here. The characteristics of the rings suggest that secondary cone degeneration is influenced by retinal position and/or other intrinsic or extrinsic factors. It has been evidenced that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model. It has been demostrated that OHT induces in the ganglion cell layer selective RGC loss that does not progress after 1 month, whereas the S- and L-cones exhibit progressive loss up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL. Palabras clave: Retina, Fotorreceptores, Conos, Retinosis Pigmentaria, Degeneración Macular Asociada a la Edad, Glaucoma. Key words: Retina, Photoreceptors, Cones, Retinitis Pgmentosa, Aging Macular Degeneration, Glaucoma.
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Bilateral early activation of retinal microglial cells in a mouse model of unilateral laser-induced experimental ocular hypertension
(Elsevier, 2018-03-09) Hoz, Rosa de; Ramírez, Ana I.; González Martín, Rosa; Ajoy, Daniel; Rojas, Blanca; Salobrar García, Elena; Valiente Soriano, Francisco J.; Avilés Trigueros, Marcelino; Villegas Pérez, María P.; Vidal Sanz, Manuel; Triviño, Alberto; Ramírez, José M.; Salazar, Juan J.; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica
The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was a significant increase in Iba1-RA. The total arbor area of microglial cells was significantly decreased in: i) OHT eyes with respect contralateral eyes and naïve-eyes in IPL; ii) OHT eyes with respect to naïve eyes in OPL. The number of microglial vertical processes connecting the OPL and OS were significantly increased in contralateral eyes compared with naïve-eyes and OHT eyes. In OPL, IPL and NFL-GCL, the cell body area of Iba-1+ cells was significantly greater in OHT eyes than in naïve and contralateral eyes, and greater in contralateral eyes than in naïve eyes. A non-proliferative microglial reactivation was detected both in contralateral eyes and in OHT eyes in an early time after unilateral laser-induced OHT (24 h). This fast microglial activation, which involves the contralateral eye, could be mediated by the immune system.
Open Access
Caracterizacion en roedores adultos de la población de células ganglionares de retina melanopsínicas y estudio de la degeneración de las células ganglionares tras hipertensión ocular y neuroprotección
(2015-07-02) Valiente Soriano, Francisco Javier; Vidal Sanz, Manuel; Agudo Barriuso, Marta; Avilés Trigueros, Marcelino; Facultad de Medicina
Objetivos • Caracterizar la población de CGRm del ratón pigmentado y albino adulto. • Estudiar la degeneración de las CGR y las CGRm después de HTO en ratón pigmentado. • Estudiar la degeneración de las CGR y las CGRm después de HTO y su protección con BDNF en rata albina adulta. Material y métodos Para la realización de los experimentos se utilizaron ratones machos pigmentados y albinos adultos y ratas hembras adultas albinas. Las manipulaciones de los animales se realizaron siguiendo la normativa europea (Directiva 2010/63/UE) y nacional (RD 53/2013) sobre la protección de los animales utilizados para la experimentación y otros fines científicos. Para caracterizar la población de CGRm del ratón pigmentado y albino adulto, se inmunodetectaron secciones transversales de retina y retinas a plano con el anticuerpo contra la melanopsina, con el anticuerpo contra Brn3a y se contratiñeron los núcleos de todas las células de la retina con DAPI. Para estudiar la proyección de las CGRm, se aplicó en ambos CS o en el muñón del nervio óptico el trazador neuronal OHSt. Para estudiar la degeneración de las CGR y las CGRm después de HTO en ratón pigmentado, se caracterizó el modelo de HTO fotocoagulando con láser las venas perilimbares y epiesclerales del ratón. Para analizar el curso temporal de daño axonal y muerte de las CGR y las CGRm, las CGR fueron trazadas retrógradamente desde los CS con OHSt, las retinas se inmunodetectaron con Brn3a y melanopsina, y se analizaron a las 2 y 4 semanas. Para estudiar la degeneración de las CGR y las CGRm después de HTO y su protección con BDNF en rata albina adulta, se analizó el curso temporal de daño axonal y muerte de las CGR y CGRm tras HTO en retinas tratadas con BDNF o vehículo. Las CGR fueron trazadas retrógradamente desde los CS con FG, y las retinas se inmunodetectaron con Brn3a y se analizaron a 12 y 15 días tras la inducción de la HTO. Para el análisis estadístico, el test Kruskal-Wallis se utilizó cuando se compararon dos o más grupos y el test Mann-Whitney cuando se compararon dos grupos solamente. Las diferencias entre grupos se consideraron estadísticamente significativas para p<0.05. Resultados Los ratones pigmentados y albinos tienen un número similar de CGRm (1.021±109 CGRm pigmentado y 962±169 CGRm albino). En los ratones pigmentados las CGRm son más abundantes en la retina tempora y en los albinos están más localizadas en la retina superior. Ambos ratones también tienen CGRm desplazadas (CGRm-d) en la capa nuclear interna, que representan el 14% del total de CGRm en ratones pigmentados y el 5% en los albinos. El marcaje desde ambos CS muestra que el 98% (pigmentado) y el 97% (albino) de la población total de CGRm se marcan retrógradamente, mientras que el estudio de colocalización de melanopsina y Brn3a confirma que un porcentaje muy pequeño de CGRm expresa este factor de transcripción en ratones. En el estudio del marcaje retrógrado colocando OHSt en el muñón del nervio óptico demuestra que no todas las CGRm eran trazadas. Existía una subpoblación de CGRm-d (14% en pigmentados y 28% en albinos) y CGRm residentes en la zona ciliar marginal de la retina (20% en pigmentados y 24% en albinos) que no se trazaban desde el nervio óptico; por lo que estas células no envían el axón a través del nervio óptico y pueden ser consideradas interneuronas de la retina, quizá relacionadas con el reflejo pupilar intrínseco. En el estudio de la caracterización del modelo de hipertensión ocular en el ratón pigmentado observamos un aumento significativo de la presión intraocular desde las primeras 6 horas de la fotocoagulación láser hasta los 5 días. En ratón pigmentado, la HTO resulta en una pérdida difusa y/o sectorial de CGR trazadas con el trazador neuronal OHSt (CGR OHSt+) (50% a 2 semanas y 62% a 4 semanas). Sin embargo, a las 2 semanas aún se observa un 66% de CGR marcadas con Brn3a (CGR Brn3a+). Esto indica que sobreviven en la retina aproximadamente un 16% de CGR cuyo transporte axonal retrógrado está comprometido. Parte de estas CGR acaban muriendo y a las 4 semanas el número de CGR trazadas con OHSt e inmunodetectadas con Brn3a se iguala. La población de CGRm disminuyó aproximadamente al 59% a las 2 semanas y al 46% a las 4 semanas, valores similares a los de las CGR Brn3a+ para los mismos tiempos. La distribución topográfica de la pérdida de CGRm, aunque era mayor en la zona supero-temporal de retina, no era sectorial, sino difusa a lo largo de la retina y no se complementaba con la distribución de la pérdida del resto de CGR. En rata albina, la HTO resulta en una pérdida sectorial de las CGR FG+ (78 y 84% a los 12 y 15 días, respectivamente). El número de CGR Brn3a+ fue significativamente mayor para ambos tiempos de estudio, esto indica que una proporción considerable (≈21 - 26%) de CGR sobreviven en la retina con su transporte axonal retrógrado deteriorado. Las CGRm también presentaron una disminución significativa (50-51%) y esta pérdida, al igual que en ratón, fue difusa. La administración intravítrea de BDNF aumentó la supervivencia de las CGR Brn3a+ a 81 y 67% a los 12 y 15 días, respectivamente, pero no tuvo ningún efecto sobre las CGRm. D. Francisco Javier Valiente Soriano “Characterization in adult rodents of the melanopsin retinal ganglion cells population and study of the retinal ganglion cells degeneration after ocular hypertension and neuroprotection” SUMMARY Objetives • Characterization of the mRGC population of adult pigmented and albino mouse. • Study the RGC and mRGC degeneration after OHT in the pigmented mouse. • Study the RGC and mRGC degeneration after OHT and their protection with BDNF in adult albino rat. Material and methods To perform the experiments we used adult pigmented mice, adult albino mice and female adult albino rats. Manipulations of animals were carried out according to the European (Directive 2010/63/UE) and National (RD 53/2013) regulations existing on the protection of animals used for experimentation and other scientific purposes. To study the characterization of the mRGC population of adult pigmented and albino mouse, cross sections of retina and whole mounts were immunoreacted with anti-melanopsin antibody, with anti-Brn3a antibody and stained with DAPI. To study the projection of the mRGC, the neuronal tracer OHSt was applied in both SC or in the optic nerve. To study the RGC and mRGC degeneration after OHT in the pigmented mouse, ocular hypertension model was performed by laser photocoagulation of the perilimbar and epiescleral veins of the experimental eye. To study the time course of the axonal damage and RGC and mRGC death caused, the RGC were traced from the SC with OHSt and retinas were immunodetected with Brn3a and melanopsin and analyzed at 2 and 4 weeks. To study the RGC and mRGC degeneration after OHT and their protection with BDNF in adult albino rat, we analyzed the time course of the axonal damage and RGC and mRGC death after OHT in BDNF or vehicle-treated retinas, RGC were retrogradely traced from the SC with the retrogradely transported tracer fluorogold (FG), retinas were immunodetected with Brn3a and analyzed at 12 and 15 days after the induction of OHT. For statistical analysis, Kruskal–Wallis test was used when comparing more than two groups and Mann–Whitney when comparing two groups only. Differences were considered significant when p<0.05. Results Both pigmented and albino mice have a similar number of mRGC (1,021±109 mRGC in pigmented, 962±169 mRGC in albino). The mRGC are most abundant in the temporal retina in pigmented mice, and in dorsal retina in albino mice. Both mice also have displaced mRGC (d-mRGC) located in the inner nuclear layer representing 14% of the total population of mRGC in pigmented mice and 5% in albino mice. The 98% (pigmented) or 97% (albino) of the total population of mRGC were marked retrogradely from both SC, while the colocalization study of melanopsin and Brn3a confirms that a very small percentage of this transcription factor was expressed by mice mRGC. A surprising fact in the study of retrograde labeling with OHSt applied on the ON stump was that not all the mRGC were traced. A subpopulation of d-mRGC (14% in pigmented and 28% in albino) and mRGC located in the ciliary marginal zone of the retina (20% pigmented and 24% in albino) that were not traced from the optic nerve, that means that these cells do not send an axon into the optic nerve and can be considered an interneuron of the retina, perhaps related to the intrinsic pupil reflex. In the study of the characterization of ocular hypertension model in pigmented mouse, a significant increase of the intraocular pressure (IOP) was observed from 6 hours of laser photocoagulation up to 5 days. OHT results in a sectorial and/or diffuse loss of RGC traced with OHSt (OHSt+RGC) (50% at 2 weeks and 62% at 4 weeks). However, at 2 weeks, 66% of RGC, which were immunodetected with Brn3a (Brn3a+RGC) were still present. This indicates that at this time around 16% of RGC survive in the retina with their retrograde axonal transport committed. These RGC, however, died at 4 weeks and the number of traced OHSt+RGC and immunodetected Brn3a+RGC was equal. The mRGC population decreased to 59% at 2 weeks and to 46% at 4 weeks. These percentages of loss were similar to the Brn3a+RGC at the same time points. The loss of the mRGC was higher in the supero-temporal area of the retina. However, this loss was not sectorial, but was diffuse along the retina, and did not parallel the distribution of loss of the rest of RGC. In albino rat, OHT resulted in a sectorial loss of FG+RGC (78-84% at 12 and 15 days, respectively). The number of Brn3a+RGC was significantly higher in both times of study, which indicates that a significant proportion (≈21-26%) of RGC survive in the retina with their impaired retrograde axonal transport. The mRGC also presented a significant reduction of approximately 50-51%, and this loss, as in mice, was diffuse. The intravitreal administration of BDNF increased the Brn3a+RGC survival to 81% and 67% at 12 and 15 days, respectively, but had no effect on the mRGC. The study of the inner retinal vasculature did not show any abnormality that could explain the sectorial loss of RGC.
Open Access
Comparative histology of pineal calcification
(Murcia : F. Hernández, 1998) Vigh, B.; Szél, A.; Debreceni, K.; Fejér, Z.; Manzano e Silva, M.J.; Vigh-Teichmann, I.
The pineal organ (pineal gland, epiphysis cerebri) contains several calcified concretions called "brain sand7' or acervuli (corpora arenacea). These concretions are conspicuous with imaging techniques and provide a useful landmark for orientation in the diagnosis of intracranial diseases. Predominantly composed of calcium and magnesium salts, corpora arenacea are numerous in old patients. In smaller number they can be present in children as well. The degree of calcification was associated to various diseases. However, the presence of calcified concretions seems not to reflect a specific pathological state. Corpora arenacea occur not only in the actual pineal tissue but also in the leptomeninges, in the habenular commissure and in the choroid plexus. Studies with the potassium pyroantimonate (PPA) method on the ultrastructural localization of free calcium ions in the human pineal, revealed the presence of calcium alongside the cell membanes, a finding that underlines the importance of membrane functions in the production of calcium deposits. Intrapineal corpora arenacea are characterized by a surface with globular structures. Meningeal acervuli that are present in the arachnoid cover of the organ, differ in structure from intrapineal ones and show a prominent concentric lamination of alternating dark and light lines. The electron-lucent lines contain more calcium than the dark ones. There is a correlation between the age of the subject and the number of layers in the largest acervuli. This suggests that the formation of these layers is connected to circannual changes in the calcium level of the organ. The histological organization of the human pineal is basically the same as that of mammalian experimental animals. Pineal concretions present in mammalian animal species are mainly of the meningeal type. Meningeal cells around acervuli contain active cytoplasmic organelles and exhibit alkaline phosphatase reaction in the rat and mink, an indication of a presumable osteoblast-like activity. Using Kossa's method for the staining of calcium deposits, a higher calcium concentration was detected in the rat pineal than in the surrounding brain tissue. Since in parathyroidectomised rats calcified deposits are larger and more numerous than in controls, the regulation of the production of acervuli by the parathyroid gland has also been postulated. In most of submammalian species, the pineal organs (pineal-, parapineal organ, frontal organ, parietal eye) are photoreceptive and organized similarly to the retina. Acervuli were found in the pineal of some birds. The pineal organs of lower vertebrates (fish, amphibians, reptiles) exhibit a high calcium content by ultrastructural calcium histochemistry (PPA-method). However, concrements are not formed. The accumulation of ca2+ seems to depend on the receptor function of the organ. Comparing pineal and retinal photoreceptors in the frog, the photoreceptor outer segments of pinealocytes as well as retinal cones and rods show a large amount of Capyroantimonate deposits. In dark adapted animals calcium ions are present in both sides of the photoreceptor membranes of the outer segment, whereas calcium is shifted extra-cellularly following light adaptation.
Open Access
Crim1–, a regulator of developmental organogenesis
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2016) Iyer, Swati; Pennisi, David J.; Piper, Michael
The regulation of growth factor localization, availability and activity is critical during embryogenesis to ensure appropriate organogenesis. This process is regulated through the coordinated expression of growth factors and their cognate receptors, as well as via proteins that can bind, sequester or localize growth factors to distinct locations. One such protein is the transmembrane protein Crim1. This protein has been shown to be expressed broadly within the developing embryo, and to regulate organogenesis within the eye, kidney and placenta. Mechanistically, Crim1 has been revealed to mediate organogenesis via its interaction with growth factors including TGFβs, BMPs, VEGFs and PDFGs. More recently, Crim1 has been shown to influence cardiac development, providing further insights into the function of this protein. This review will provide an overview of the role of Crim1 in organogenesis, largely focusing on how this protein regulates growth factor signaling in the nascent heart. Moreover, we will address the challenges ahead relating to further elucidating how Crim1 functions during development.
Open Access
Current knowledge of dystrophin and dystrophin-associated proteins in the retina
(Murcia : F. Hernández, 2000) Ueda, H.; Baba, T.; Ohno, S.
Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (RMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and B-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains fulllength dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). B-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.
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Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains
(Elsevier, 2016-01-12) De Groef, Lies; Dekeyster, Eline; Geeraerts, Emiel; Lefevere, Evy ; Stalmans, Ingeborg; Moons, Lieve; Salinas Navarro, Manuel Ángel; Anatomía Humana y Psicobiología
Mouse disease models have proven indispensable in glaucoma research, yet the complexity of the vast number of models and mouse strains has also led to confusing findings. In this study, we evaluated baseline intraocular pressure, retinal histology, and retinofugal projections in three mouse strains commonly used in glaucoma research, i.e. C57Bl/6, C57Bl/6-Tyrc, and CD-1 mice. We found that the mouse strains under study do not only display moderate variations in their intraocular pressure, retinal architecture, and retinal ganglion cell density, also the retinofugal projections to the dorsal lateral geniculate nucleus and the superior colliculus revealed striking differences, potentially underlying diverging optokinetic tracking responses and visual acuity. Next, we reviewed the success rate of three models of (glaucomatous) optic neuropathies (intravitreal N-methyl-d-aspartic acid injection, optic nerve crush, and laser photocoagulation-induced ocular hypertension), looking for differences in disease susceptibility between these mouse strains. Different genetic backgrounds and albinism led to differential susceptibility to experimentally induced retinal ganglion cell death among these three mouse strains. Overall, CD-1 mice appeared to have the highest sensitivity to retinal ganglion cell damage, while the C57Bl/6 background was more resistant in the three models used.
Open Access
Efecto a corto y largo plazo de diferentes períodos de isquemia transitoria en la supervivencia de las células ganglionares de la retina de rata adulta: estudio cuantitativo
(2012-12-21) Sellés Navarro, Inmaculada; Vidal Sanz, Manuel; Villegas Pérez, María Paz; Miralles de Imperial Mora-Figueroa, Jaime; Departamentos y Servicios::Departamentos de la UMU::Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica
La retina es un tejido nervioso que parece tener una mayor tolerancia ante situaciones de hipoxia que el resto del sistema nervioso central (SNC). Sin embargo, las consecuencias funcionales derivadas de la privación de flujo sanguíneo son graves y en la mayoría de los casos irreversibles. Se han realizado numerosas investigaciones con el fin de conocer las alteraciones morfológicas y funcionales de la retina ante situaciones de isquemia de diferente intensidad, sin embargo se carecen de … [+]estudios que caractericen específicamente el comportamiento de las células ganglionares de la retina (GCR) ante la isquemia utilizando métodos que nos permiten cuantificar la muerte de las (GCR). Queremos determinar su grado de tolerancia ante la isquemia, y estudiar el patrón de muerte de estas células tras someterlas a diferentes periodos de isquemia transitoria basándonos en técnicas neuroanatómicas. Hemos marcado las CGR con un trazador neuronal fluorescente, el fluoro-Gold, que aplicado en los principales núcleos de proyección de la vía óptica de la rata adulta nos ha permitido distinguir y diferenciar las CGR
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Effects of different neurotrophic factors on the survival of retinal ganglion cells after a complete intraorbital nerve crush injury: a quantitative in vivo study
(Elsevier, 2009-06-15) Parrilla Reverter, Guillermo; Agudo, Marta; Sobrado Calvo, Paloma; Villegas Pérez, María P.; Vidal Sanz, Manuel; Salinas Navarro, Manuel Ángel; Anatomía Humana y Psicobiología
We examined in adult Sprague Dawley rats the loss of retinal ganglion cells (RGCs) induced by complete intraorbital optic nerve crush (IONC) as well as the effects of several neurotrophic factors to prevent IONC-induced RGC loss. Completeness of the IONC lesion was assessed by investigating the orthograde and retrograde transport of neuronal tracers applied to the origin and termination of the retinotectal pathway. RGC survival after IONC alone or combined with intraocular injection of the neurotrophic factors NT-4, BDNF or CNTF was quantified at survival intervals ranging from 5 to 12 days post-lesion (dpl) by identifying RGCs that had been pre-labelled with fluorogold (FG). RGC loss first appeared at 7 dpl and by 12 dpl only 32% of the RGC population remained in the retina. Intraocular administration of NT-4, BDNF or CNTF resulted in almost a complete protection against IONC-induced RGC loss by 7 dpl, and the protection remained significant by 12 dpl only for NT-4 and BDNF. We have analyzed these results taking into account our previous studies on the loss of RGCs induced by intraorbital optic nerve transection (IONT) and concluded that RGC loss induced by IONC is slower and less severe than that following IONT. Moreover, as for IONT-induced RGC loss, IONC-induced RGC loss may also be prevented with administration of NT-4, BDNF or CNTF, though for NT-4 and CNTF their neuroprotective effects differ depending on the injury type. Overall this data underscore the importance of the type of ON injury on the pattern of RGC degeneration as well as in their response to neuroprotective treatments.
Open Access
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
(2017-07-19) Di Pierdomenico, Johnny; Villegas Pérez, María Paz; García Ayuso, Diego; Agudo Barriuso, Marta; Facultad de Medicina
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.
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Extent and duration of recovered pupillary light reflex following retinal ganglion cell axon regeneration through peripheral nerve grafts directed to the pretectum in adult rats
(Elsevier, 1998-12) Whiteley, S. J. O.; Sauvé, Y.; Avilés Trigueros, Marcelino; Vidal Sanz, Manuel; Lund, R. D.; Oftalmología, Optometría, Otorrinolaringología y Anatomía Patológica
The functional reinnervation of the olivary pretectal nucleus (OPN) was studied in adult rats with peripheral nerve (PN) grafts bridging the interrupted retinopretectal pathway. Functional recovery was assessed quantitatively using established pupillometry techniques. The effect of intravitreal tuftsin fragment 1–3 (tuftsin 1–3) injections during the grafting procedure was also studied. A total of 53 adult rats received autologous PN grafts connecting the ocular stump of the transected optic nerve to the ipsilateral OPN. The contralateral eye was enucleated to remove the input from that eye to the OPN. A pupillary light reflex was elicited from 35 of the 53 PN-grafted animals and in the best cases, a response was obtained which compared closely to that recorded from control animals. Tuftsin 1–3 was found to increase the rate of recovery of the response. The response amplitude of PN-grafted rats was generally found to diminish with repeated stimulus presentation and also appeared to deteriorate with age. This was in contrast to control animals' responses. However, a PLR could still be elicited in 3 of the 6 animals studied 15 months after PN-grafting. These findings indicate that a near-normal PLR function can be restored using a peripheral nerve graft, but there are a number of factors that are likely to compromise optimal outcome.
Open Access
Fenómenos asociados a la difusión de la luz en visión e imágenes de la retina
(2016-09-05) Christaras, Dimitrios; Artal Soriano, Pablo; Facultad de Medicina
Cuando la luz viaja a través del ojo para formar la imagen retiniana se ve afectada por los medios ópticos de forma que el objeto y la imagen nunca pueden ser idénticos. La luz se dispersa según una función, característica de cada sistema óptico, la función de dispersión de punto (PSF) que depende básicamente de tres fenómenos: la difracción, las aberraciones y el scattering, cada uno afectando la PSF de un modo distinto. La difracción, aunque en realidad es un fenómeno general que describe el cambio de dirección de la luz cuando encuentra un obstáculo físico, en la óptica fisiológica se refiere a la difracción de la luz en la pupila. Las aberraciones, por otro lado, describen cualquier desviación del sistema óptico perfecto, o cualquier desviación de la aproximación paraxial. En las últimas dos décadas, las aberraciones oculares de bajo y alto orden se han medido de manera efectiva y se han corregido con éxito utilizando la óptica adaptativa. Tanto la difracción como las aberraciones, afectan principalmente a la parte central de la PSF, que está relacionada con las características de alta frecuencia, o los detalles de la imagen. La difracción y las aberraciones degradan la imagen de la retina que afecta la agudeza visual y la sensibilidad al contraste. El scattering se refiere a la dispersión de la luz debido a las estructuras de aproximadamente 10 micrómetros o menos, y afecta no solo a la zona central, pero a la zona periférica de la PSF también. Por lo tanto, contrariamente a la difracción y las aberraciones, los efectos de la dispersión en la imagen afectan igual a las frecuencias más bajas, y se percibe como una pérdida de contraste que depende en gran medida del rango dinámico de la imagen, o de la presencia de fuentes de resplandor (¨glare sources¨). El scattering aumenta con la edad y especialmente en los pacientes con cataratas y puede conducir a una pérdida severa de contraste. La PSF afecta de la misma manera a la visión y a la formación de imágenes del fondo del ojo, a pesar de que la retina y los procesos que tienen lugar en nivel neural puedan compensar parcialmente los errores de la visión. El objetivo de esta tesis es estudiar el efecto del scattering en la visión y en la obtención de imágenes de la retina. El primer paso fue construir un modelo teórico del fondo de ojo y simular, usando técnicas Monte Carlo, la difusión de la luz en el fondo del ojo con diferentes longitudes de onda, y su dependencia con la pigmentación. Se demostró que existe una fuerte dependencia de la difusión en el fondo del ojo con la longitud de onda, con una reflectancia aproximadamente 10 veces más alta en longitudes de onda más largas. Además, se observó una dependencia de la difusión del fondo de ojo en los cambios con la pigmentación, únicamente para las longitudes de onda más largas, mientras que las longitudes de onda medianas no se vieron afectados. Además, la difusión en el fondo se comparó con el scattering producido en la óptica del ojo y el rango espacial de cada fenómeno. El estudio mostró que para longitudes de onda intermedias la difusión es dominante hasta aproximadamente 2 grados de ángulo visual, mientras que para las longitudes de onda más largas pueden dominar hasta los 4 grados. Se investigó el efecto de la difusión del fondo de ojo en la visión. En primer lugar, usando una técnica óptica, se cuantificó la luz difundida en el fondo del ojo para diferentes condiciones espaciales en verde (550 nm) y rojo (650 nm) midiendo la reflectancia del fondo de ojo para 6 sujetos agrupados según su pigmentación. Se encontró un aumento significativo en la reflectancia en los sujetos con más pigmentación para las longitudes de onda más largas, en línea con las simulaciones y estudios anteriores. En segundo lugar, se construyó un sistema psicofísico, basado en el método de Heterochromatic Flicker Photometry (HFP), para medir la sensibilidad en verde y rojo para dos condiciones espaciales distintas. Las medidas ópticas se compararon con las medidas psicofísicas donde se observó un aumento de la sensibilidad con la reflectancia del fondo de ojo, consistentes en todos los sujetos. Dado que la reflectancia del fondo de ojo depende de la pigmentación, los individuos de pigmentación baja muestran una sensibilidad al color rojo mayor. En la otra parte de la tesis, se estudió el scattering en los medios oculares, centrando en el efecto de la dispersión en la imagen del fondo. Para este propósito, un sistema óptico se construyó, basado en el principio de doble paso, capaz de adquirir imágenes del fondo ocular a diferentes longitudes de onda y tamaños de campo. El método completo se probó y validó primero en una configuración en paso simple, donde el scattering se introdujo por medio de filtros. La reconstrucción de la PSF completa se realizó en 7 sujetos utilizando el método de integración óptica suponiendo una PSF basada en la CIE glare function. Se registraron imágenes del fondo a diferentes longitudes de onda y la imagen original se reconstruyó aplicando un método de ¨enhancing¨ de contraste usando con la PSF reconstruida. Se demostró que la compensación de scattering puede mejorar significativamente el contraste de la imagen. El nivel de mejora depende no sólo de la cantidad de scattering para la longitud de onda específica, sino también del rango dinámico de la imagen del fondo. Se encontró una mejora de alrededor del 10 % para la configuración específica. El método de compensación de scattering se aplicó en el caso de la medida de la densidad óptica del pigmento macular (MPOD) usando imágenes retinianas. Según este método se tomaron dos imágenes de fondo de ojo, una a la longitud de onda donde el pigmento macular no absorbe y otra a una longitud de onda en el que muestra su pico de absorción y se extrajo la MPOD usando una fórmula matemática. El cálculo se hizo antes y después de aplicar el algoritmo de compensación de scattering para 6 sujetos y se midió una subestimación media de la MPOD de aproximadamente un 10 %. Por último, para probar los resultados con un método ya conocido de MPOD se construyó un sistema adicional psicofísico, basado en el método de HFP, que está afectado únicamente a un pequeño grado de scattering. Se observó una buena correlación entre los dos métodos que aumenta cuando el scattering se compensa. Summary When light travels through the eye to form the retinal image, it is affected by the optical media in a number of ways, such that the object and image space can never be identical. The light is spread according to a function, characteristic of the system, called the Point Spread Function (PSF) and it depends on three main phenomena: diffraction, aberrations and scattering, each one of them with different effects. Diffraction, although in reality a very general effect describing essentially the redirection of the light when meeting an obstacle, in physiological optics usually refers to diffraction at the pupil. %This simplifies the description of the phenomenon, making it possible to approximate its effect with a simple formula. Aberrations, on the other hand, is a more general term describing any deviation from the perfect model, or any deviation from the paraxial approximation. In the last two decades, low and high order ocular aberrations have been successfully measured and corrected using adaptive optics. Both diffraction and aberrations affect mainly the central part of the PSF, that is related to the high frequency features, or the details of the image. Diffraction and aberrations degrade the retinal image affecting visual acuity and contrast sensitivity. Scattering refers to the spreading of the light due to structures of about 10 microns or less, and it is not restricted to the central part of the PSF but affects its wide-angle parts as well. Therefore, contrary to diffraction and aberrations, scattering also affects low frequencies and it is perceived as a loss of contrast which depends heavily on the dynamic range of the image, or the presence of glare sources. Scattering increases with age and especially in cataract patients and can lead to a severe contrast loss. The PSF affects in exactly the same way, both vision and fundus imaging, although the retina and the processes taking place at a neural level can partially compensate the errors for vision. The purpose of this thesis is to study the effect of scattering in vision and in fundus imaging. The first step was to build a theoretical fundus model and quantify light diffusion in the fundus at different wavelengths and its dependence on the melanin pigmentation, simulating realistic pigmentation values found in a normal population. It was shown that there is a strong dependence of fundus diffusion on wavelength, with even 10 times higher reflectance or more at longer wavelengths. Moreover, a dependence of fundus diffusion was observed on changes in melanin pigmentation but only for longer wavelengths, whereas medium wavelengths were unaffected. Additionally, fundus diffusion was compared to scattering in the optical media and the spatial dominance of each phenomenon was studied. The study showed that for medium wavelengths diffusion is dominant to up to about 2 degrees of visual angle, whereas for longer wavelengths the domain of dominance increases to 4 degrees. The effect of fundus diffusion in vision was investigated. Firstly, using an optical technique, diffused light in the fundus was quantified for different spatial conditions at green (550nm) and red (650nm) through the measurement of fundus reflectance for 6 subjects grouped according to their melanin pigmentation. There was a significant increase in reflectance at light pigmentation subjects for longer wavelengths, which was in line with previous simulations and studies. Secondly, a psychophysical setup based on the Heterochromatic Flicker Photometry (HFP) method was built and the green to red color sensitivity for two different spatial conditions was measured. The measurements of the optical system were then compared to the psychophysical ones where an increase in sensitivity with fundus reflectance was observed, consistent in all subjects. Consequently, since fundus reflectance depended on pigmentation, light pigmentation individuals have an increased red to green color sensitivity. In the other part of the thesis, the focus was shifted towards the study of scattering in the ocular media, with the center of attention being the effect of scattering in fundus imaging. For this purpose, an optical setup was built based on the double-pass principle, capable of acquiring fundus images at various wavelengths and field sizes. The complete method was first tested and validated in a single-pass setup where scattering was introduced by a scatter filter. The reconstruction of the wide angle PSF was done for 7 subjects using the method of optical integration assuming a PSF based on the CIE glare function. Fundus images at different wavelengths were acquired for the subjects and the original intensity map of the image was reconstructed by applying a custom contrast enhancement method using the reconstructed PSF. It was shown that this scattering compensation technique can significantly improve the contrast of the image. The improvement depended not only on the amount of scatter for the specific wavelength but also on the dynamic range of the fundus. An improvement of about 10% was found for the specific setup. The scattering compensation method was applied in the case of the Macular Pigment Optical Density (MPOD) measurement using reflectometry. In this method two fundus images were taken, one at wavelength where macular pigment does not absorb and another at a wavelength where it shows its peak absorption and the MPOD was extracted using the pixel intensities of the two images. The calculation was done both before and after applying the scattering compensation algorithm for 6 subjects and an average MPOD underestimation of about 10% was observed. Finally, in order to test the results against a proven MPOD measurement, a psychophysical setup was built, based on the HFP method, which is affected only to a small degree by scattering. A very good correlation between the two methods was observed and the agreement increased when scattering was compensated.
Open Access
Fine structure of the retina and pigment epithelium in the creek chub, Semotilus atromaculatus (Cyprinidae, Teleostei)
(Murcia : F. Hernández, 1996) Collin, S.P.; Collin, H.B.; Ali, M.A.
The structure of the light- and dark-adapted retina, the pigment epithelium and the choroid of the creek chub, Semotilus atromaculatus (Cyprinidae, Teleostei) is examined by light and electron microscopy. An extensive network of vitreal blood vessels emanating from the hyaloid artery enters the eye with the optic nerve and overlies the inner limiting membrane. This membrane closely apposes the fine protrusions of the Müller cell processes which traverse the entire retina, dividing the inner retina into alternating fascicles of ganglion cells and optic axons. The inner nuclear layer consists of bipolar, amacrine, Müller cell soma and two layers of horizontal cells. The outer plexiform layer possesses both rod spherules and cone pedicles. Each rod spherule consists of a single synaptic ribbon in either a triad or quadrad junctional arrangement within the invaginating terminal endings of the bipolar and horizontal cell processes. In contrast, cone pedicles possess multiple synaptic ribbons within their junctional complexes and, in the light-adapted state, the horizontal cell processes show spinule formation. Four photoreceptor types are identified on morphological criteria; unequal double cones, large single cones, small single cones and rods. Al1 but the small single cones are capable of retinomotor responses. The rod to cone ratio is approximately 5:l and the rods form two ill-defined rows in the light-adapted condition. The retinal pigment epithelium possesses two types of osmiophilic granules. These are bound within slender microvilli and migrate vitread to surround the photoreceptors in response to light. Bruch's membrane is trilaminar and the vascularised choroid consists of up to three layers of melanocytes. The endothelial borders of the choroidal blood vessels abutting the outer lamina of Bruch's membrane are fenestrated.
Open Access
Fine structure of the retina of black bass, Micropterus salmoides. Centrarchidae, Teleostei
(Murcia : F. Hernández, 1999) García, M.; De Juan, J.
The structure of light- and dark-adapted retina of the black bass, Micropterus salmoides has been studied by light and electron microscopy. This retina lacks blood vessels at all levels. The optic fiber layer is divided into fascicles by the processes of Miiller cells and the ganglion cell layer is represented by a single row of voluminous cells. The inner nuclear layer consists of two layers of horizontal cells and bipolar, amacrine and interplexiform cells. In the outer plexiform layer we observed the synaptic terminals of photoreceptor cells, rod spherules and cone pedicles and terminal processes of bipolar and horizontal cells. The spherules have a single synaptic ribbon and the pedicles possess multiple synaptic ribbons. Morphologically, we have identified three types of photoreceptors: rods, single cones and equal double cones which undergo retinomotor movements in response to changes in light conditions. The cones are arranged in a square mosaic whereas the rods are dispersed between the cones.
Open Access
Fine structure of the retinal photoreceptors of the barred owl (Strix varia)
(Murcia : F. Hernández, 1996) Braekevelt, Charlie R.; Smith, S.A.; Smith, B.J.
The photoreceptors of the barred owl (Strix varia) consist of rods, single cones and unequal double cones present in a ratio of about 35:1:3. In the lightadapted condition the rods are of uniform diameter along their entire length and are therefore not felt to undergo photomechanical changes. The rod outer segment consists of a stack of scalloped bimembranous discs enclosed in a limiting membrane. The rod inner segment displays an ellipsoid of mitochondria, much rough endoplasmic reticulum (RER), numerous polysomes, Golgi zones and autophagic vacuoles, but no hyperboloid of glycogen. Single cones show a slightly tapered outer segment and a heterogeneous oil droplet along with an ellipsoid of mitochondria at the apex of the inner segment. Double cones consist of a larger chief member which also displays a heterogeneous oil droplet and a slightly smaller accesory member which does not. Both members of the double cone as well as the single cones show plentiful polysomes and RER as well as Golgi zones in the inner segment, but none of the cones possessed a condensed paraboloid of glycogen. The contiguous membranes of the chief and accessory cones displayed a few presumed junctional complexes. Judging by their elongated shape in the light-adapted state, cones in this species do not undergo retinomotor movements. Rods and both types of cones have both invaginated (ribbon) and superficial (conventional) synaptic sites.
Open Access
Identificación y caracterización de la población total de las células ganglionares de la retina en rata : nuevos métodos de trazado, expresión de melanopsina y de factores de transcripción Brn3:estudio de la respuesta neuronal y microglial a la axotomía y efecto del envejecimiento en la retina
(2015-12-01) Nadal-Nicolás, Francisco Manuel; Agudo Barriuso, Marta; Vidal Sanz, Manuel; Facultad de Medicina
Introducción La retina es parte del sistema nervioso central (SNC) y se localiza en la cara interna del globo ocular. Su función principal es la fototransducción de las ondas electromagnéticas del espectro de la luz visible en energía eléctrica. Esta función es realizada por los fotorreceptores (conos y bastones). Tras su procesamiento, la información llega a las células ganglionares de retina (CGR). Las CGR son las únicas neuronas aferentes de la retina y transmiten la información visual al cerebro a través de sus axones, que forman el nervio óptico (NO). En roedores, la mayoría de las CGR proyecta contralateralmente, siendo la población ipsilateral menor del 5%. Dentro de las CGR existe un subtipo que contiene un pigmento fotosensible, la melanopsina, que les confiere la propiedad de la fototransducción. Estas CGR melanopsínicas son responsables principalmente de funciones extravisuales o no formadoras de imágenes, como son el reflejo pupilar o la sincronización del ritmo circadiano con la luz. Objetivos 1º Caracterizar un muevo marcador para identificar las CGR de rata: Brn3a. 2º. Caracterizar la expresión de los factores de transcripción de familia Brn3 en las CGR de rata: Brn3a, Brn3b y Brn3c. 3º. Caracterizar las CGR desplazadas en rata albina y pigmentada. 4º. Caracterizar la población de CGR con proyección retino-retiniana en rata. 5º. Desarrollar nuevos métodos de trazado de las CGR de rata: desde el nervio óptico intacto y desde el tracto óptico. 6º. Analizar el efecto del trazado y la lesión axonal en las CGR melanopsínicas y en la expresión de melanopsina en rata albina y pigmentada. 7º. Caracterizar el efecto a largo plazo de la lesión del nervio óptico en la población general de CGR y CGR melanopsínicas ortotópicas y desplazadas y en el resto de las células que componen la capa de células ganglionares en rata albina y pigmentada. 8º. Caracterizar la respuesta de las células microgliales en la retina de rata tras axotomía del nervio óptico. 9º. Caracterizar el efecto del envejecimiento en la retina de rata albina y pigmentada. Material y Métodos. Resultados y Conclusiones Para identificar las CGR clásicamente se han usado técnicas de trazado. Los trazadores como el Fluorogold® (FG) se aplican o en el NO, para identificar la proyección retinofugal completa, o en los colículos superiores (CS), adonde proyectan el 98,4%% de las CGRs en roedores. En esta tesis hemos puesto a punto dos métodos nuevos de trazado: desde el NO intacto o desde el tracto óptico mediante una inyección bilateral estereotáctica. Ambas técnicas son asequibles, reproducibles y fiables. Además, hemos caracterizado el Brn3a como marcador de CGR. El Brn3a es un marcador fiable y eficiente para identificar y cuantificar las CGR en retinas intactas y con lesión axonal y además, permite analizar la topografía de las CGR después del daño axonal ya que a diferencia de los trazadores neuronales, no presenta interferencia con la microglía fagocítica trazada transcelularmente. Conjuntamente hemos analizado la expresión de los tres miembros de la familia Brn3 en las CGR, y hemos demostrado que el 70% de las CGR co-expresan dos o tres miembros de la familia Brn3 y el 30% restante expresa solamente Brn3a (26%) o Brn3b (4%) en retina de rata. Por tanto, el Brn3a se expresa en todas las CGRs exceptuando las CGR melanopsínicas y la mitad de la población ipsilateral. La mayoría de las CGR se localizan en la capa de las células ganglionares (CCG), conocidas como CGR ortotópicas (CGRo), aunque una pequeña población de CGR se encuentra desplazada a la capa nuclear interna o a la capa plexiforme interna. Estas CGR se llaman células de Dogiel o CGR desplazadas (CGRd). Nosotros hemos estudiado a ambas poblaciones en paralelo. Mientras que las CGR ortotópicas se distribuyen principalmente por la región dorso-central de la retina, las CGR desplazadas tienen una topografía diferente, se encuentran en el ecuador de la retina, con un densidad mayor en la retina temporal y son más abundantes en la rata pigmentada. La mayoría de las CGRd expresan Brn3a, y una pequeña proporción expresa melanopsina, estas últimas se distribuyen de manera similar a las CGRo melanopsínicas: son más abundantes en la retina dorso-temporal. Existe una pequeña población de CGR que proyecta a la retina contralateral, éstas son las CGR de proyección retino-retiniana (CGR ret-ret). Hemos corroborado que esta proyección es mayor en animales jóvenes que en adultos y que se encuentran preferentemente en la retina nasal y, además hemos demostrado que éstas expresan Brn3a o melanopsina y que, las que lo hacen, son las que se mantienen en los animales adultos. En la CCG, además de las CGRo hay otras poblaciones celulares: células endoteliales, células gliales y las células amacrinas desplazadas (CAd). En esta tesis hemos descrito que el 45% de las neuronas de la CCG son CGRs y el 55% restante son CAd. Y si excluimos las células endoteliales, las células gliales representarían un 10% de la población total de la capa de células ganglionares. En esta tesis, también analizamos como el albinismo afecta a las CGR. El albinismo es una enfermedad hereditaria, en la que hay una ausencia parcial o total de pigmentación que, entre otras, provoca una serie de anomalías en el sistema visual tales como una menor proyección ipsilateral y número de CGRd y, la agudeza visual y el nistagmus optocinético están afectados. Nosotros hemos demostrado que el albinismo produce los mismos defectos en la población melanopsínica que en el resto de las CGR: disminución en el número de CGRd-m y una proporción inferior de ipsilateralidad. Las lesiones en el SNC provocan la muerte neuronal con secuelas permanentes e irrecuperables, ya que las neuronas del SNC no se reemplazan. En esta tesis hemos utilizado dos modelos de degeneración de SNC que afectan específicamente a las CGR: la lesión traumática axonal por sección (SNO) o aplastamiento (ApNO) de nervio óptico, y la hipertensión ocular (HTO) como modelo de glaucoma aumentando la presión intraocular por fotocoagulación láser de la malla trabecular y las venas perilimbares y episclerales. Usando el modelo de la elevación de la presión intraocular hemos observado que las CGR desplazadas presentan la misma respuesta que las CGR ortotópicas. Y los modelos de axotomía de nervio óptico también nos han permitido documentar que estas lesiones causan la pérdida específica de CGR (ortotópicas y desplazadas), sin afectar a otras poblaciones de la capa de células ganglionares. Sin embargo, dentro de las CGR, las CGR melanopsinicas tienen un curso temporal de pérdida diferente, son más resistentes a la lesión, pero la expresión de melanopsina se infra-regula transitoriamente como respuesta tanto la axotomía como al trazado retrógrado desde el NO. Así, este hallazgo debe ser tenido en cuenta cuando se utilice la melanopsina para estudiar la población de las CGR intrínsicamente fotosensibles. Las células de la microglía (CM) son los macrófagos residentes del SNC. En condiciones normales se encuentran en estado de vigilancia. Sin embargo, tras una lesión neurodegenerativa se activan fagocitando desechos celulares. La aproximación experimental que se utiliza para identificar las CM que han fagocitado una neurona (o CM fagocíticas, CMF) se basa en el hecho de que las CM acumulan en sus fagolisosomas productos exógenos, proceso conocido como marcaje transcelular. Así, cuando una CM fagocita una CGR en degeneración previamente trazada, acumula el trazador siendo posible distinguirla de las CM que no han fagocitado. En esta tesis hemos cuantificado y analizado la distribución de las CM en la CCG y la CPI tanto en animales intactos como después de ambos modelos de axotomia (SNO y ApNO). La aparición de las CMF después del insulto aumenta al aumentar el tiempo post-lesión y se distribuyen en la región central de la retina donde hay una mayor pérdida de las CGR, a diferencia de los animales intactos donde se distribuyen homogéneamente. Aunque éste aumento de CMF es más rápido después de la SNO, existe una correlación lineal y topográfica entre la aparición de las CMF y la pérdida de CGR. La aparición de las CMF en la CCG y el descenso de las CM no fagocíticas en la CPI a 14d de ambas lesiones, sugiere que tras la lesión de las CGR las CM migran entre ambas capas. La pérdida funcional o estructural de la actividad sensorial relacionada con la edad, es muy relevante cuando afecta al sistema visual, ya que de él dependemos más que de otros sentidos. No sólo los componentes puramente físicos implicados en la visión (córnea, cristalino, humor vítreo y humor acuoso) sufren cambios estructurales que influyen en la eficiencia de la transmisión lumínica perjudicando la calidad de la visión, sino que hay pérdida numérica y funcional en las poblaciones celulares implicadas en la transmisión de la información hasta el cerebro que aumenta con la edad. En esta tesis hemos comprobado que el envejecimiento causa principalmente un déficit funcional de la retina en ambas estirpes de rata analizadas. Pero anatómicamente, ni el número de células en la CCG, ni el transporte axonal anterógrado disminuyen con la edad, solamente en la cepa pigmentada, hay un descenso del número de fotoreceptores tipo cono. Y mediante un análisis “in vivo” (SD-OCT), también hemos observado un alargamiento y adelgazamiento progresivo de la retina. Para la realización de esta tesis ha sido necesario el desarrollo de rutinas informáticas que permitan tanto la cuantificación como la representación grafica de la distribución de las diversas poblaciones celulares estudiadas en la retina. Todas estas metodologías automáticas fueron realizadas en colaboración con D. Manuel Jiménez López. Introduction The retina is part of the central nervous system (CNS) and it is located in the posterior part of the ocular globe. The main function of the retina is to sense light. Photoreceptors, cones and rods and send the luminous information to retinal ganglion cells (RGCs) through intermediate neurons. RGCs are the only afferent retinal neurons and transmit this information from the retina to the retinorecipient areas in the brain through their axons that form the optic nerve (ON). In rodents, the majority of RGC project to the contralateral superior colliculi (SCi), being the ipsilateral projection smaller than 5%. There is a subtype of RGC that expresses a photosensitive pigment, melanopsin, that confers them the ability of phototransduction. Melanopsin+RGC (m+RGC) are responsible for the non visual functions triggered by light, such as the pupilary reflex and the circadian photoentrainment. Objetives 1st. To characterize Brn3a as a marker of rat RGCs. 2nd. To characterize the expression of Brn3 transcription factors, Brn3a, Brn3b and Brn3c, in rat RGCs. 3rd. To characterize the population of displaced RGCs in albino and pigmented rats. 4th. To characterize the population of RGCs that project retino-retinially. 5th. To investigate the efficiency of two new methods to trace rat RGCs: from the intact optic nerve and from the optic tract. 6th. To analyze the effect of tracing or axotomy on the expresión of melanopsin and detection of melanopsin+RGCs in albino and pigmented rat RGCs. 7th. To analyze in albino and pigmented rats the long term effect of optic nerve injury on RGCs and m+RGCs orthotopic and displaced, and on the rest of the ganglion cell layer cells. 8th. To analyze the microglial response in the rat retina after optic nerve axotomy. 9th. To study in albino and pigmented rats the effect of aging on the retina. Material and Methods. Results and Conclusions Retrograde tracing with tracers such as Fluorogold® (FG) is the classical approach to identify RGCs. Tracers are applied in the ON to identify the whole retinofugal projection or on the SC, where 98.4% of the RGC project to. In these thesis, we have tuned up two new methods to trace rat RGCs: from the intact optic nerve and from the optic tract by a bilateral stereotactic injection. Both techniques are affordable, reproducible and reliable. In addition we have characterized the Brn3a as a marker of rat RGCs. Brn3a is a reliable marker to identify, quantify and assess the viability of rat RGCs in health and disease. In addition, Brn3a immunodetection allows quantifying and determining the topography of RGCs after a given injury without interference of transcellularly- labelled microglial cells. We have analyzed the expression of the three members of the Brn3 family RGC, and we have shown that 70% of RGC co-express two or three Brn3 members and the remaining 30% expresses only Brn3a (25%) or Brn3b (4%). Brn3a is expressed by all RGCs except melanopsin+ ones and half of the ipsilateral projection. Most of the RGC are placed in the ganglion cell layer (GCL), these are orthotopic RGC (oRGC). However a small proportion of them is located in the inner nuclear layer or in the inner plexiform layer. These are known as Dogiel's cells or displaced RGC (dRGC). We have studied both counterpart together. While the ortothopic RGCs, are denser in the dorso-central retina, the displaced RGCs have a different topography, they are found in the retinal equator, with a higher density in the temporal retina and are more abundant in pigmented animals. Most of the dRGCs express Brn3a, and a small proportion express melanopsin, the last ones have a similar distribution than the m+-oRGCs: they are more abundant in the dorso-temporal retina. There is a small number of RGC projects to the contralateral retina, these are retino-retinal projecting RGC (ret-ret RGC). We have beared out the retino-retinal projection is minute but higher in young than in adult animals. Ret-ret RGCs are mainly nasal, and express Brn3a or melanopsin and those do it, are preserved in adult animals. In the GCL besides oRGC there are endothelial cells, glial cells and displaced amacrine cells (dAC). In this work, we have described that 45%percent of neurons in the GCL are RGCs, and 55% are displaced amacrine cells. And, if we exclude the endothelial cells, glial cells represent 10% of the total cell population of the ganglion cell layer. In this thesis, we have also analyzed how albinism affects RGCs. Albinism is a hereditary disease caused by the partial or total lack of pigmentation that causes a long list of abnormalities in the visual system such as an impaired visual acuity and optokinetic nystagmus and defects in the crossing of the retinofugal projections. Here, we added to this knowledge, that albinism produces in the melanopsin population the same defects than in the general RGC population: reduced number of displaced m+RGCs and a lower ipsilaterally. CNS lesions induce the permanent and irreversible death of the affected neurons, since CNS neurons do not proliferate and thus, are not replaced. In this thesis we have used two models of RGC degeneration: traumatic axonal injury (axotomy) transecting (ONT) or crushing (ONC) the optic nerve, and ocular hypertension (OHT), a model of glaucoma created by increasing the intraocular pressure with laser photocoagulation of the trabecular meshwork, the perilimbar and episcleral veins. Using the ocular hypertension, we have observed that the dRGCs and oRGC respond similarly to lesion. And the axotomy models also allowed us to document that after axotomy only RGCs are lost (ortothopic and displaced) without affecting other populations in the ganglion cell layer. However, within RGCs, the m+RGCs have a different course of loss, they are more resistant to injury, but the expression of melanopsin is temporarily under-regulated in response to both axotomy and retrograde tracing from the NO. Thus, this finding should be considered when melanopsin is used to study the population of the intrinsically photosensitive CGR. Microglial cells (MC) are the CNS resident macrophages. In the healthy CNS they are found in a resting (surveying) state. However, during a neurodegenerative process, they activate and among other functions, phagocytose the cellular debris. The experimental approach to identify CM that have phagocytose a neuron (phagocytic microglial cells, PMC) is based on the fact that CM accumulate in their phagolysosomes exogenous compounds, such as tracers. This process is known as transcellular tracing. Thus, when a MC engulfs a traced RGC it is possible to distinguish it from the rest of MC. In this thesis, we quantified and analyzed the distribution of MC in the GCL and the IPL in intact animal or after both axotomy models (SNO and APNO). The number of CMF after the insult increases with time post-injury. These PMC are distributed in the central region of the retina where the loss of RGCs is greater, unlike in intact animals where they are homogeneously distributed. The appearance of PMC correlates linearly and topographically with the loss of RGCs. The increase of PMC in the GCL and the decrease of MC in the IPL suggest that upon RGC injury, MC migrate between both layers. Aging is very relevant when affects the visual system, since we depend on vision more than on any other senses. Not only the physical components of the eye (cornea, lens, vitreous and aqueous humor) through which the light passes age, there is also a progressive neuronal loss and degeneration. In this thesis we found that aging causes, mainly, a functional deficit in the retina in both rat strains. Anatomically, neither the number of cells in the GCL nor the anterograde axonal transport diminishes with age, however, in the pigmented, but not in the albino rat, there is a loss of cone photoreceptors. And by “in vivo” analysis (SD-OCT), we have also observed a progressive elongation and thinning of the retina. For the consecution of this thesis it has been necessary to develop several automated routines to perform the quantification and graphic representation of the different cell populations analyzed. All these automated methodologies were created in collaboration with D. Manuel Jimenez López.
Open Access
Identification of protein kinase C α- and tyrosine hydroxylase-immunoreactive cells in the microbat retina
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2018) Park, Eun Bee; Jeon, Joo Yeong; Jeon, Chang Jin
A growing number of studies have revealed the functional neuroarchitecture of the microbat retina and suggested that microbats can see using their eyes. To better understand the organization of the microbat retina, quantitative analysis of protein kinase C alpha (PKCα)- and tyrosine hydroxylase (TH)-immunoreactive (IR) cells was conducted on the greater horseshoe bat (Rhinolophus ferrumequinum) retina. As a result, PKCα immunoreactivity was observed in rod bipolar cells, consistent with previous studies on other mammalian retinas. PKCα-IR cell distribution in the inner nuclear layer showed regional differences in density, with the highest density found in the nasal retina. The average density of PKCα-IR cells was 10,487±441 cells/mm2 (mean ± SD; n=4), with a total of 43,077±1,843 cells/retina. TH-IR cells in the Rhinolophus ferrumequinum retina could be classified into four types based on soma location and ramification in the inner plexiform layer: conventional amacrine, displaced amacrine, interplexiform, and intercalated cells. The majority of TH-IR cells were conventional amacrine cells. TH-IR cells were nonrandomly distributed at low density over the retina. The average density was 29.7±3.1 cells/mm2 (mean ± SD; n=3), with a total of 124.0±11.3 cells/retina. TH-IR processes showed varicosities and formed ring-like structures encircling AII amacrine cells. Our study provides the foundation for understanding the neurochemical architecture of the microbat retina and supports the notion that the eyes do play a role in the visual system of microbats.