Glia-Retinal Ganglion Cell Interactions ins the Mammalian Retina: A Neuroprotetive Approach.

191 p.Las células ganglionares de la retina (RGCs) son las neuronas responsables de la comunicación entre el ojo y el cerebro, y su muerte puede causar una ceguera irreversible, como ocurre en el glaucoma. Las RGCs se encuentran en estrecho contacto con las células de la glía. En la retina de mamíferos hay tres tipos de células gliales, cuya función es mantener la homeostasis de la retina, estos tipos son: astrocitos, células Müller y microglía. Cuando se induce un daño o lesión en la retina, la glía puede percibir este daño y responder a él, pudiendo actuar como sensores de daño además de poder neuroproteger a las RGCs.A fin de estudiar la relación entre la glía y las RGCs, en la presente Tesis hemos utilizado un modelo de hipoxia neonatal en cerdos. Hemos encontrado que el cerebro percibe el daño antes que la retina, así los astrocitos y las neuronas en colículo superior se ven dañadas antes que los astrocitos y las RGCs en la retina. Este hecho podría explicarse debido la presencia de células Müller en la retina.Para estudiar la neuroprotección de las RGCs por las células de Müller y la relación entre estos dos tipos celulares hemos utilizado cultivos primarios. Las células Müller pueden neuroproteger a las RGCs por contacto célula-célula, además de secretar moléculas con efecto neuroprotectoras. En la presente Tesis hemos analizando el secretoma de las células de Müller mediante proteómica, combinado con una estrategia funcional en la que se analiza la supervivencia y neuritogénesis de RGCs. Tras el análisis hemos seleccionado varias moléculas candidatas y hemos comprobado que la osteopontina y la basigina son proteínas candidatas noveles que aumentan la supervivencia de las RGCs.Hemos comprobado que el plasma rico en factores de crecimiento (PRGF), al contrario que en otros tipos celulares, disminuye drásticamente la supervivencia de las RGCs, aumenta la proliferación de las células de Müller y activa la respuesta inflamatoria en la retina, observado como un aumento de la migración de microglía, que puede ser debido a la presencia de citoquinas inflamatorias en el PRGF.Sabiendo que la osteopontina tiene propiedades neuroprotectoras, estudiamos el efecto de su ausencia in vivo. La falta de osteopontina en ratones knock-out produce la muerte de las células ganglionares de la retina, así como la disminución de astrocitos, confirmando que la osteopontina es importante para el funcionamiento de la retina y podría ser un buen candidato para tratar la neurodegeneración retiniana.Finalmente, la osteopontina también puede usarse como biomarcador de daño, debido a su sobreexpresión tras una lesión, como es el pinzamiento del nervio óptico, donde sus niveles de RNA aumentan más de 9 veces en la cabeza del nervio óptico. Con el fin de establecer un buen biomarcador molecular de daño de las células ganglionares, nos propusimos cuantificar la sobreexpresión de la osteopontina, además de la lipocalina 2, a nivel proteico tanto en la cabeza del nervio óptico como en el humor acuoso. Dichos estudios aun se encuentran en progreso.En conclusión, la glía retiniana puede ayudarnos a detectar signos de daño mediante cambios morfológicos o mediante la secreción de marcadores moleculares. Además, podemos usar sus propiedades neuroprotectoras para desarrollar posibles tratamientos contra enfermedades neurodegenerativas en las que se afectan las células ganglionares de la retina

Immunohistochemical Characterisation of the Whale Retina

The eye of the largest adult mammal in the world, the whale, offers a unique opportunity to study the evolution of the visual system and its adaptation to aquatic environments. However, the difficulties in obtaining cetacean samples mean these animals have been poorly studied. Thus, the aim of this study was to characterise the different neurons and glial cells in the whale retina by immunohistochemistry using a range of molecular markers. The whale retinal neurons were analysed using different antibodies, labelling retinal ganglion cells (RGCs), photoreceptors, bipolar and amacrine cells. Finally, glial cells were also labelled, including astrocytes, Müller cells and microglia. Thioflavin S was also used to label oligomers and plaques of misfolded proteins. Molecular markers were used to label the specific structures in the whale retinas, as in terrestrial mammalian retinas. However, unlike the retina of most land mammals, whale cones do not express the cone markers used. It is important to highlight the large size of whale RGCs. All the neurofilament (NF) antibodies used labelled whale RGCs, but not all RGCs were labelled by all the NF antibodies used, as it occurs in the porcine and human retina. It is also noteworthy that intrinsically photosensitive RGCs, labelled with melanopsin, form an extraordinary network in the whale retina. The M1, M2, and M3 subtypes of melanopsin positive-cells were detected. Degenerative neurite beading was observed on RGC axons and dendrites when the retina was analysed 48 h post-mortem. In addition, there was a weak Thioflavin S labelling at the edges of some RGCs in a punctuate pattern that possibly reflects an early sign of neurodegeneration. In conclusion, the whale retina differs from that of terrestrial mammals. Their monochromatic rod vision due to the evolutionary loss of cone photoreceptors and the well-developed melanopsin-positive RGC network could, in part, explain the visual perception of these mammals in the deep sea

Immunohistochemical Characterisation of the Whale Retina

[EN] The eye of the largest adult mammal in the world, the whale, offers a unique opportunity to study the evolution of the visual system and its adaptation to aquatic environments. However, the difficulties in obtaining cetacean samples mean these animals have been poorly studied. Thus, the aim of this study was to characterise the different neurons and glial cells in the whale retina by immunohistochemistry using a range of molecular markers. The whale retinal neurons were analysed using different antibodies, labelling retinal ganglion cells (RGCs), photoreceptors, bipolar and amacrine cells. Finally, glial cells were also labelled, including astrocytes, Muller cells and microglia. Thioflavin S was also used to label oligomers and plaques of misfolded proteins. Molecular markers were used to label the specific structures in the whale retinas, as in terrestrial mammalian retinas. However, unlike the retina of most land mammals, whale cones do not express the cone markers used. It is important to highlight the large size of whale RGCs. All the neurofilament (NF) antibodies used labelled whale RGCs, but not all RGCs were labelled by all the NF antibodies used, as it occurs in the porcine and human retina. It is also noteworthy that intrinsically photosensitive RGCs, labelled with melanopsin, form an extraordinary network in the whale retina. The M1, M2, and M3 subtypes of melanopsin positive-cells were detected. Degenerative neurite beading was observed on RGC axons and dendrites when the retina was analysed 48 h post-mortem. In addition, there was a weak Thioflavin S labelling at the edges of some RGCs in a punctuate pattern that possibly reflects an early sign of neurodegeneration. In conclusion, the whale retina differs from that of terrestrial mammals. Their monochromatic rod vision due to the evolutionary loss of cone photoreceptors and the well-developed melanopsin-positive RGC network could, in part, explain the visual perception of these mammals in the deep sea

How Do Whales See?

The eyes of two whales Balaenoptera physalus and Baleoptera borealis were studied by our group. In this chapter, we present the anatomical, histological, immunohistochemical and ultrastructural studies of the eyes of both types of whales. Based on the results, we can conclude that at least in these two species, the whales are rod monochromat; their resolution is very limited due to the reduced number of retinal ganglion cells, some of which were giant size (more than 100 micrometers in diameter). The excellent representation of melanopsinic positive retinal ganglion cells suggests an adaptation to the dim light as well as involvement in the circadian rhythms. The large cavernous body located in the back of the eye may provide a mechanism that allows them to move the eye forward and backwords; this may facilitate focusing and provide protection from cold deep-sea temperatures

Differential Distribution of RBPMS in Pig, Rat, and Human Retina after Damage

RNA binding protein with multiple splicing (RBPMS) is expressed exclusively in retinal ganglion cells (RGCs) in the retina and can label all RGCs in normal retinas of mice, rats, guinea pigs, rabbits, cats, and monkeys, but its function in these cells is not known. As a result of the limited knowledge regarding RBPMS, we analyzed the expression of RBPMS in the retina of different mammalian species (humans, pigs, and rats), in various stages of development (neonatal and adult) and with different levels of injury (control, hypoxia, and organotypic culture or explants). In control conditions, RBPMS was localized in the RGCs somas in the ganglion cell layer, whereas in hypoxic conditions, it was localized in the RGCs dendrites in the inner plexiform layer. Such differential distributions of RBPMS occurred in all analyzed species, and in adult and neonatal retinas. Furthermore, we demonstrate RBPMS localization in the degenerating RGCs axons in the nerve fiber layer of retinal explants. This is the first evidence regarding the possible transport of RBPMS in response to physiological damage in a mammalian retina. Therefore, RBPMS should be further investigated in relation to its role in axonal and dendritic degeneration.This research was funded by ELKARTEK KK-2019/00086, Research groups of the UPV/EHU (GIU 2018/50)and MINECO-Retos (PID2019-111139RB-I00) to E.V. Programa de perfeccionamiento de personal InvestigadorDoctor, Gobierno Vasco to X.P

Characteristics of Whale Muller Glia in Primary and Immortalized Cultures

[EN] Muller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Muller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Muller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Muller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Muller glia to neurons. Here, the Muller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Muller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Muller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Muller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Muller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular markers of Muller cells, with an increase in dedifferentiation markers like alpha-SMA and beta-III tubulin, while they preserve their GS expression depending on the culture passage. Here we also discuss the possible influence of the animal's age and size on these cells, and on their senescence.This study was supported by ELKARTEK (KK-2019/00086), MINECO-Retos (PID2019-111139RB-I00), Grupos UPV/EHU (GIU 2018/150), and Proyectos de Investigación Básica y/o Aplicada (PIBA_2020_1_0026) to EV, Basque Government postdoctoral grant (POS_2020_2_0031) to XP, UPV/EHU- Bordeaux predoctoral grant (PIFBUR20/10) to SB, and UPV/EHU postdoctoral grant (ESPDOC20/058) to NR

Dexamethasone Protects Retinal Ganglion Cells But Not Muller Glia Against Hyperglycemia In Vitro

Diabetic retinopathy (DR) is a common complication of diabetes, for which hyperglycemia is a major etiological factor. It is known that retinal glia (Muller cells) and retinal ganglion cells (RGCs) are affected by diabetes, and there is evidence that DR is associated with neural degeneration. Dexamethasone is a glucocorticoid used to treat many inflammatory and autoimmune conditions, including several eye diseases like DR. Thus, our goal was to study the effect of dexamethasone on the survival of RGCs and Muller glial cells isolated from rat retinas and maintained in vitro under hyperglycemic conditions. The behavior of primary RGC cell cultures, and of mixed RGC and Muller cell co-cultures, was studied in hyperglycemic conditions (30 mM glucose), both in the presence and absence of Dexamethasone (1 mu M). RGC and Muller cell survival was evaluated, and the conditioned media of these cultures was collected to quantify the inflammatory cytokines secreted by these cells using a multiplex assay. The role of IL-1 beta, IL-6 and TNF alpha in RGC death was also evaluated by adding these cytokines to the co-cultures. RGC survival decreased significantly when these cells were grown in high glucose conditions, reaching 54% survival when they were grown alone and only 33% when co-cultured with Muller glia. The analysis of the cytokines in the conditioned media revealed an increase in IL-1 beta, IL-6 and TNF alpha under hyperglycemic conditions, which reverted to the basal concentration in co-cultures maintained in the presence of dexamethasone. Finally, when these cytokines were added to co-cultures they appeared to have a direct effect on RGC survival. Hence, these cytokines could be implicated in the death of RGCs when glucose concentrations increase and dexamethasone might protect RGCs from the cell death induced in these conditions.This work was funded by the support of Retos-MINECO Fondos Feder (RTC-2016-48231) and Grupos Consolidados del Gobierno Vasco (IT437-10) to E.V

The Effect of Plasma Rich in Growth Factors on Microglial Migration, Macroglial Gliosis and Proliferation, and Neuronal Survival

Plasma rich in growth factors (PRGF) is a subtype of platelet-rich plasma that has being employed in the clinic due to its capacity to accelerate tissue regeneration. Autologous PRGF has been used in ophthalmology to repair a range of retinal pathologies with some efficiency. In the present study, we have explored the role of PRGF and its effect on microglial motility, as well as its possible pro-inflammatory effects. Organotypic cultures from adult pig retinas were used to test the effect of the PRGF obtained from human as well as pig blood. Microglial migration, as well as gliosis, proliferation and the survival of retinal ganglion cells (RGCs) were analyzed by immunohistochemistry. The cytokines present in these PRGFs were analyzed by multiplex ELISA. In addition, we set out to determine if blocking some of the inflammatory components of PRGF alter its effect on microglial migration. In organotypic cultures, PRGF induces microglial migration to the outer nuclear layers as a sign of inflammation. This phenomenon could be due to the presence of several cytokines in PRGF that were quantified here, such as the major pro-inflammatory cytokines IL-1beta, IL-6 and TNFalpha. Heterologous PRGF (human) and longer periods of cultured (3days) induced more microglia migration than autologous porcine PRGF. Moreover, the migratory effect of microglia was partially mitigated by: 1) heat inactivation of the PRGF; 2) the presence of dexamethasone; or 3) anti-cytokine factors. Furthermore, PRGF seems not to affect gliosis, proliferation or RGC survival in organotypic cultures of adult porcine retinas. PRGF can trigger an inflammatory response as witnessed by the activation of microglial migration in the retina. This can be prevented by using autologous PRGF or if this is not possible due to autoimmune diseases, by mitigating its inflammatory effect. In addition, PRGF does not increase either the proliferation rate of microglial cells or the survival of neurons. We cannot discard the possible positive effect of microglial cells on retinal function. Further studies should be performed to warrant the use of PRGF on the nervous systemWe acknowledge the support of MINECO-Retos Fondos Fender (RTC-2016-48231), Gobierno Vasco (PUE_2018_1_0004), ELKARTEK (KK-2019/00086), MINECO-Retos (PID2019-111139RB-I00) and PIBA (2020-1-0026) to E

The Extracellular Matrix of the Human and Whale Cornea and Sclera: Implications in Glaucoma and Other Pathologies

The cornea is the transparent part of the eye that allows light to enter into the eye and reach the retina, thereby activating the neurons that will send messages to the brain. The sclera is the hard-white part of the eye, and its main function is to provide structure and form to the eye, and to support the retina. Indeed, while the cornea best performs its main functions when transparent and it is capable of adapting its curvature to allow the eye to focus, the sclera must be opaque and hard to function correctly. Both structures are mainly composed of collagen, some elastic fibres and ground substance, all components of the Extracellular Matrix. The disposition of the collagen fibres and the amount of ground substance around the fibres is responsible for the differences in the aspect of both these structures. In this chapter, for the first time we have compared the structure and ultrastructure of the cornea and sclera in humans and the whale adult (18mts) Balaenoptera physalus, the second largest animal on the planet. We will discuss how the differences in their structure may be related to the maintenance of intraocular pressure in their distinct environments, which is of particular clinical interest as increased intraocular pressure is one of the main causes underlying the development of open angle glaucoma

Plasma Rich in Growth Factors (PRGF) Increases the Number of Retinal Muller Glia in Culture but Not the Survival of Retinal Neurons

Plasma rich in growth factors (PRGF) is a subtype of platelet-rich plasma (PRP) that stimulates tissue regeneration and may promote neuronal survival. It has been employed in ophthalmology to achieve tissue repair in some retinal pathologies, although how PRGF acts in the retina is still poorly understood. As a part of the central nervous system, the retina has limited capacity for repair capacity following damage, and retinal insult can provoke the death of retinal ganglion cells (RGCs), potentially producing irreversible blindness. RGCs are in close contact with glial cells, such as Muller cells, that help maintain homeostasis in the retina. In this study, the aim was to determine whether PRGF can protect RGCs and whether it increases the number of Muller cells. Therefore, PRGF were tested on primary cell cultures of porcine RGCs and Muller cells, as well as on co-cultures of these two cell types. Moreover, the inflammatory component of PRGF was analyzed and the cytokines in the different PRGFs were quantified. In addition, we set out to determine if blocking the inflammatory components of PRGF alters its effect on the cells in culture. The presence of PRGF compromises RGC survival in pure cultures and in co-culture with Muller cells, but this effect was reversed by heat-inactivation of the PRGF. The detrimental effect of PRGF on RGCs could be in part due to the presence of cytokines and specifically, to the presence of pro-inflammatory cytokines that compromise their survival. However, other factors are likely to be present in the PRGF that have a deleterious effect on the RGCs since the exposure to antibodies against these cytokines were insufficient to protect RGCs. Moreover, PRGF promotes Muller cell survival. In conclusion, PRGF hinders the survival of RGCs in the presence or absence of Muller cells, yet it promotes Muller cell survival that could be the reason of retina healing observed in the in vivo treatments, with some cytokines possibly implicated. Although PRGF could stimulate tissue regeneration, further studies should be performed to evaluate the effect of PRGF on neurons and the implication of its potential inflammatory role in such processesWe acknowledge the support of MINECO-Retos Fondos Fender (RTC-2016-48231), Gobierno Vasco (PUE_2018_1_0004), ELKARTEK (KK-2019/00086), PIBA 2020-1-0026 and MINECO-Retos (PID2019-111139RB-I00) to E