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

[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

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

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

Comparative study of the lipid profile of tears and plasma enriched in growth factors.

[EN] The Tear Film Lipid Layer (TFLL) acts primarily as an interface between the aqueous layer and air. Tear film lipid is composed of a thin layer of polar lipids that interact with the secretory layer of the underlying mucosa and a thicker layer of non-polar lipids at the air interface. The tear film has a complex structure and composition that protects the cornea, promotes wound healing, and maintains high-quality vision. Plasma Rich in Growth Factor (PRGF) eye drops emerged as an exciting new treatment for corneal epitheliopathies, including aqueous deficient dry eye. The purpose of this study was to compare the lipidomic profile of eye drops obtained from PRGF with tear lipidome to determine whether PRGF drops could be an adequate complement to tears in patients with impaired TFLL. To address this study, tears and blood was collected and processed from healthy donors to obtain PRGF eye drops. Samples were aliquoted and stored at -80°C until use. The lipid profiles of these samples were analysed by Ultrahigh Performance Liquid Chromatography (UHPLC) using a Vanquish UHPLC system to obtain untargeted lipidome profiles on a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. In PRGF eye drops, 408 lipids were identified in ESI+mode and 183 in ESI- mode, and they were grouped into 15 different lipid classes from four distinct categories. By contrast, 112 lipid species were identified from tear samples in ESI+mode and 36 in ESI- mode, belonging to 12 lipid classes from six different categories. The relative abundance of most lipid species was much greater in the PRGF eye drops than in the tear, although there were some lipids present in tears that were not found in the PRGF, such as wax esters and (O-acyl)-omega-hydroxy fatty acids. In summary, these results suggest that the lipids present in PRGF eye drops could serve as a tear supplement in individuals in whom tear lipid composition is altered, although there are differences in the lipid profile of these two fluids.The authors wish to acknowledge the financial support received to carry out this work from: MINECO-Retos Fondos Fender (RTC-2016-48231), Gobierno Vasco (PUE_2018_1_0004), ELKARTEK (KK-2019/00086), PIBA 2020-1-0026, MINECO-Retos (PID2019-111139RB-I00), ELKARTEK (KK-2021-00023) to EV and FISS-21-RD21/0002/0041 to AA

Comparative lipidomic analysis of mammalian retinal ganglion cells and Muller glia in situ and in vitro using High-Resolution Imaging Mass Spectrometry

In order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Muller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Muller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Muller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Muller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Muller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value <= 0.05) between the areas containing Muller glia and RGCs. Likewise, different densities of lipids were evident between the RGC and Muller glia cultures in vitro. Finally, a comparative analysis of the lipid profiles in the retinal sections and microarrays identified six peaks that corresponded to a collection of 10 lipids characteristic of retinal cells. These lipids were identified by MS/MS. The analyses performed on the RGC layer of the retina, on RGCs in culture and using cell membrane microarrays of RGCs indicate that the lipid composition of the retina detected in sections is preserved in primary cell cultures. Specific lipid species were found in RGCs and Muller glia, allowing both cell types to be identified by a lipid fingerprint. Further studies into these specific lipids and of their behavior in pathological conditions may well help identify novel therapeutic targets for ocular diseases.This study was supported by the grants RETOS MINECO FEDER (RTC-2016-48231), PUE 2018-1-0004, UPV/EHU PPGA 18/18 and Elkartek KK-2019/00086 to E.V

Potential Tear Biomarkers for the Diagnosis of Parkinson’s Disease—A Pilot Study

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In this study, the tear proteome profile of patients with idiopathic PD (iPD, n = 24), carriers of the E46K-SNCA mutation (n = 3) and healthy control (CT, n = 27) subjects was analyzed to identify candidate biomarkers for the diagnosis of PD. An observational, prospective and case-control pilot study was carried out, analyzing the participants tear samples by nano-liquid chromatography–mass spectrometry (nLC–MS/MS) and assessing their neurological impairment. The proteomic data obtained are available at ProteomeXchange with identifier 10.6019/PXD028811. These analyses led to the identification of 560 tear proteins, some of which were deregulated in PD patients and that have been implicated in immune responses, inflammation, apoptosis, collagen degradation, protein synthesis, defense, lipid transport and altered lysosomal function. Of these proteins, six were related to neurodegenerative processes and showed a good capacity to classify patients and controls. These findings revealed that certain proteins were upregulated in the tears of PD patients, mainly proteins involved in lysosomal function. Thus, in this study, tear proteins were identified that are implicated in neurodegeneration and that may be related to an aggressive disease phenotype in PD patients.This work was supported by MINECO-Retos Fondos Fender (RTC-2016-48231), Gobierno Vasco (PUE_2018_1_0004), ELKARTEK (KK-2019/00086), PIBA 2020-1-0026, MINECO-Retos (PID2019-111139RB-I00) and ELKARTEK (KK-2021/00023)