Macroglial Cells Research Articles

A1 adenosine receptor evoked Ca2+ signals in astrocytes

Abstract In addition to neurons, glial cells make up the nervous system. These glial cells are divided into macroglial cells - including astrocytes - and microglial cells. Astrocytes have several important functions in the central nervous system, such as synthesizing transmitters and releasing them, thereby activating the surrounding neurons and astrocytes. Astrocytes do not generate action potentials, but they can transmit information to neighboring cells and react to external stimuli by employing Ca2+ dynamics. Increased neuronal activity can lead to an increased extracellular level of adenosine, reflected by a change in the metabolic state. Adenosine is a naturally occurring nucleoside distributed throughout the body as a metabolic intermediary. Its action is mediated by binding to adenosine receptors, such as the G-protein coupled receptors (GPCRs) A1 adenosine receptor. The binding of adenosine to the specific receptor type activates different signaling pathways in astrocytes. In our work, we studied Ca2+ signals generated upon adenosine A1 receptor activation by adding the pathway to the computational astrocyte model by Oschmann et.al (2017). We investigated the influence of the high-affinity A1 adenosine receptor in various astrocyte domains on the regulated Ca2+ release from the endoplasmic reticulum (ER) of astrocytes and concluded that the receptor-dependent pathway contributes to Ca2+ signals mainly in the soma.

Wound Healing in a Porcine Model of Retinal Holes.

To investigate retinal wound healing, we created a new porcine model of retinal hole and identified the cells involved in hole closure. Sixteen landrace pigs underwent vitrectomy, and a subretinal bleb was created before cutting a retinal hole using a 23G vitrector. No tamponade was used. Before surgery and one, two, and four weeks after surgery, the eyes were examined by optical coherence tomography and color fundus photos. At the end of follow-up, the eyes were enucleated for histology. Tissue sections of 5 µm were prepared for hematoxylin-eosin staining and immunohistochemical analysis with antibodies to retinal glial and epithelial cells. Retinal holes below 1380 µm in diameter closed spontaneously within four weeks, whereas larger holes remained open. Hole closure was mediated by central movement of the edges of the hole and in most cases the formation of a gliotic plug. Fluorescence microscopy revealed that the plug consisted of cells positive for glial fibrillary acidic protein, indicating the presence of macroglial cell types. Specifically, the plug was positive for S100 calcium-binding protein B, mainly representing astrocytes, while it was negative for anti-glutamine syntethase, representing Müller glia. These findings suggest that astrocytes are the predominating cell type in the plug. Minimal glial reaction was seen in the retinal holes that did not close. We present a new porcine model for investigating large retinal holes. The retinal holes closed by approximation of hole edges, and the remnant retinal defect was closed with an astroglial plug.

Reconstruction of macroglia and adult neurogenesis evolution through cross-species single-cell transcriptomic analyses.

Macroglia fulfill essential functions in the adult vertebrate brain, producing and maintaining neurons and regulating neuronal communication. However, we still know little about their emergence and diversification. We used the zebrafish D. rerio as a distant vertebrate model with moderate glial diversity as anchor to reanalyze datasets covering over 600 million years of evolution. We identify core features of adult neurogenesis and innovations in the mammalian lineage with a potential link to the rarity of radial glia-like cells in adult humans. Our results also suggest that functions associated with astrocytes originated in a multifunctional cell type fulfilling both neural stem cell and astrocytic functions before these diverged. Finally, we identify conserved elements of macroglial cell identity and function and their time of emergence during evolution.

Cilastatin as a Potential Anti-Inflammatory and Neuroprotective Treatment in the Management of Glaucoma.

Glaucoma is a neurodegenerative disease that causes blindness. In this study, we aimed to evaluate the protective role of cilastatin (CIL), generally used in the treatment of nephropathologies associated with inflammation, in an experimental mouse model based on unilateral (left) laser-induced ocular hypertension (OHT). Male Swiss mice were administered CIL daily (300 mg/kg, i.p.) two days before OHT surgery until sacrifice 3 or 7 days later. Intraocular Pressure (IOP), as well as retinal ganglion cell (RGC) survival, was registered, and the inflammatory responses of macroglial and microglial cells were studied via immunohistochemical techniques. Results from OHT eyes were compared to normotensive contralateral (CONTRA) and naïve control eyes considering nine retinal areas and all retinal layers. OHT successfully increased IOP values in OHT eyes but not in CONTRA eyes; CIL did not affect IOP values. Surgery induced a higher loss of RGCs in OHT eyes than in CONTRA eyes, while CIL attenuated this loss. Similarly, surgery increased macroglial and microglial activation in OHT eyes and to a lesser extent in CONTRA eyes; CIL prevented both macroglial and microglial activation in OHT and CONTRA eyes. Therefore, CIL arises as a potential effective strategy to reduce OHT-associated damage in the retina of experimental mice.

Inflammatory response of both retinas and superior colliculi to unilateral optic nerve axotomy

Objective: Unilateral optic nerve crush (ONC) induces a bilateral inflammatory reaction affecting both injured and contralateral un‐injured retinas. However, few works have investigated the spread of inflammation along visual pathway. Our aim was to study the glial response and the underlying molecular changes in both retinas and the superior colliculi (SCi), the main projection areas of retinal ganglion cells in mice. In this species, >90% of RGCs project to the contralateral SC (cSCi).Methods: The left optic nerve of adult pigmented C57Bl/6 male mice were crushed (ONC) at 0.5 mm from the optic disk or underwent surgery without crushing (Sham). One group of intact mice was used as controls. Two studies were carried out:1) At 1, 3, 9 and 30 days post lesion (dpl) both retinas and SCi were freshly dissected and their RNA extracted. Using qPCR we measured the expression levels of TGF‐β1, IL‐1β, TNF‐α, Iba1, GFAP, MHC II and TSPO in retinas and SCi. In SCi extracts we also studied Casp3, CXCR1, Lcn2, IL‐6, CD206, IL‐4, and AQ4. Hptr and Gapdh were used as houskeepings. Intact retinas and SCi were used as controls.2) Three, 9 and 30 days after ONC or sham‐surgery immunodetection of microglia and macroglial cells was carried out in sagittal brain sections. The area occupied by GFAP and MHC II signal as well as the density of Iba1+ and CD68+ cells was quantified in both SCi. Intact brains were used as controls.Results: Sham surgery alone modified gene expression in both retinas and SCi compared to intact animals. Therefore, to isolate the effect of ONC, we compared ONC vs. Sham. After ONC, TNF‐α upregulated early in both retinas and IL‐β remained overexpressed in injured retina at day 30. GFAP overexpression was exclusively observed in injured retina while Iba1 overexpression was presented in both. TSPO and MHCII and Casp3 overexpression was observed only in the cSC.Anatomical microglial activation (rounded somas and CD68 expression) was observed in the cSC from day 3 onwards after ONC. In the ipsilateral SC (iSC) microglial cells did not show signs of activation. Astrocytes hypertrophied in both SCi from day 9 after ONC, but this response was also observed in Sham‐animals.Conclusions: Sham surgery promotes an inflammatory and glial response that must be considered in experimental designs. Unilateral ONC triggers a bilateral response which extends anterogradely and retrogradely along the visual pathway.

Differences in Müller's glia between the central and peripheral retina and its effect in glaucoma

Glaucoma is a neurodegenerative disease and the leading cause of irreversible blindness in the world, whose main risk factor is increased intraocular pressure. When visual problems are first detected in patients with glaucoma, already half of the retinal ganglion cells (RGCs), the neurons that send visual information from the eye to the brain, have died principally in the peripheral retina. The timeframe of cell death is prolonged, lasting even decades after an initial diagnosis. Müller glia (MG) are the main macroglial cell in the retina, play prominent roles regulating normal and pathological physiology and react to neuronal injury in glaucoma. The change to a reactive phenotype of MG initiates signalling cascades that may serve a neuroprotective role, but may also proceed to promote damaging effects on RGCs. However, the underlying mechanisms and signalling pathways that specifically promote protective versus destructive roles of reactive glial cells are mostly unclear.Using cell cultures of Müller cells, retinal ganglion cells or cocultures subjected to hydrostatic pressure, we have been able to prove that Müller cells in the periphery react to pressure by secreting harmful proteins that induce ganglion cell death. Proteomic analysis has provided us with information on the different behaviour of Müller cells in the periphery versus those in the center.Elevated hydrostatic pressure on MG induces the expression of proteins associated to apoptosis, oxidative stress and inflammation in the peripheral MG. This study confirms that MG could have a role in RGCs death in glaucoma opening new avenues for research. Preventing MG response to elevated pressure, MG can neuroprotect RGCs in early stages of glaucoma.Supported by ELKARTEK (KK‐2019/00086), MINECO‐Retos (PID2019‐111139RB‐I00), PIBA (2020_1_0026), Grupos Consolidados Gobierno Vasco, UPV/EHU:

Sufficient Cav-1 levels in the endothelium are critical for the maintenance of the neurovascular unit in the retina

BackgroundCaveolin-1 (Cav-1) is a pivotal protein in the plasma membrane. Studies on homozygous Cav-1 deficient mice revealed that Cav-1 is essential for endothelial function and angiogenesis in the retina. However, whether a reduction in Cav-1 content hampers the neurovascular unit (NVU) in the retina is unclear. Thus, this study examines the NVU in the retinas of heterozygous Cav-1 deficient (Cav-1+/−) mice and analyzes possible underlying mechanisms.MethodsThe vascular, glial and neuronal components in the retina were evaluated using retinal morphometry, whole mount retinal immunofluorescence staining, histological analysis and optical coherence tomography. In addition, immunoblotting and immunofluorescence staining, subcellular fractionation, biotin labeling of cell surface proteins, and proximity ligation assay were employed to detect expression and localization of proteins in the retina or endothelial cells (ECs) upon knockdown of Cav-1 with Cav-1 siRNA.ResultsCav-1+/− retinas showed a significant reduction in pericyte coverage along with an increase in acellular capillaries compared to controls at 8 months of age, but not at 1 month. A significant loss and obvious morphological abnormalities of smooth muscle cells were observed in 8-month-old Cav-1+/− retinal arterioles. Macroglial and microglial cells were activated in the Cav-1+/− retinas. A transient significant delay in retinal angiogenesis was detected in Cav-1+/− retinas at p5, which was however no longer detectable at p10. The Cav-1+/− retinas displayed increased vascular permeability and a notable reduction in VEGFR2 content at 8 months. In vitro, siRNA-mediated knockdown experiments in ECs revealed that the loss of Cav-1 in ECs resulted in decreased levels of VEGFR2, VE-Cadherin and their interaction at the plasma membrane as well.ConclusionOur results indicate that a sufficient Cav-1 level over 50% of its normal abundance is vital for the proper localization of VEGFR2 and VE-cadherin, likely in a complex, at the plasma membrane, which is essential for the maintenance of normal NVU in the retina.

Participation of cAMP-Mediated Signaling Transduction in the Regulation of the Secretory Function of Neuroglia during Ethanol-Induced Neurodegeneration.

We studied the involvement of cAMP and PKA in the regulation of the secretion of neurotrophic growth factors by macro-and microglial cells in the model of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP in the secretion of neurotrophins by intact astrocytes and oligodendrocytes was shown, while PKA does not participate in this process. On the contrary, the inhibitory role of cAMP (implemented via PKA activation) in the production of neurogenesis stimulators by microglial cells under conditions of optimal vital activity was found. Under the influence of ethanol, the role of cAMP and PKA in the production of growth factors by macroglial cells was considerably changed. The involvement of PKA in the cAMP-dependent signaling pathways and inversion of the role of this signaling pathway in the implementation of the neurotrophic secretory function of astrocytes and oligodendrocytes, respectively, directly exposed to ethanol in vitro were noted. Long-term exposure of the nervous tissue to ethanol in vivo led to the loss of the stimulating role of cAMP/PKA signaling on neurotrophin secretion by macroglial cells without affecting its inhibitory role in the regulation of this function in microglial cells.

Dysfunction of NG2 glial cells affects neuronal plasticity and behavior.

NG2 glia represents a distinct type of macroglial cells in the CNS and is unique among glia because they receive synaptic input from neurons. They are abundantly present in white and gray matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of gray matter NG2 glia and their synaptic input are still ill defined. Here, we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Kir4.1 was deleted at postnatal day 23-26 (recombination efficiency about 75%) and mice were investigated 3-8 weeks later. Notably, these mice with dysfunctional NG2 glia demonstrated improved spatial memory as revealed by testing new object location recognition while working and social memory remained unaffected. Focussing on the hippocampus, we found that loss of Kir4.1 potentiated synaptic depolarizations of NG2 glia and stimulated the expression of myelin basic protein while proliferation and differentiation of hippocampal NG2 glia remained largely unaffected. Mice with targeted deletion of the K+ channel in NG2 glia showed impaired long-term potentiation at CA3-CA1 synapses, which could be fully rescued by extracellular application of a TrkB receptor agonist. Our data demonstrate that proper NG2 glia function is important for normal brain function and behavior.

Comparative Analysis of Retinal Organotypic Cultures and In Vivo Axotomized Retinas

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury.

Müller glial cell photosensitivity: A novel function bringing higher complexity to vertebrate retinal physiology

The retina of vertebrates is responsible for detecting and capturing ambient light for image and non-image forming (NIF) functions through diverse projections to the brain which regulate visual processing, pupillary light responses, photic synchronization of circadian rhythms and suppression of pineal melatonin, among others. For this, vertebrates have retained through evolution at least two sets of photoreceptors specialized primarily in such visual and NIF tasks: visual photoreceptors cones and rods responsible for day/night vision, and intrinsically photosensitive retinal ganglion cells (ipRGC) together with horizontal cells in some vertebrates, expressing melanopsin (Opn4). Interestingly, Opn4 as well as encephalopsin (Opn3) and neuropsin (Opn5), responding to blue and UV light, respectively, are expressed in the inner retina and command light detection in the blue range of the visible spectra; they are responsible for a number of NIF functions still lacking characterization. Though most retinal photoreceptors are derived from ciliary or neuronal progenitor cells, in recent years Müller glial cells (MCs), the most abundant retinal glial cell type, have been shown to express different blue opsins (Opn3 and Opn5) and the photoisomerase retinal G protein-coupled receptor (RGR), and to respond directly to light. MCs display different essential functions to maintain the homeostasis and cell survival of the whole retina, contributing to glutamate metabolism and chromophore recycling. The novel photoreceptive capacity of MCs, mainly in the blue region, offers several highly intriguing possibilities that increase the complexity levels for light detection in the retina and its light-activated circuits, calling for further investigation. The goal of the present review is to discuss the state of the art of research on the principal macroglial cells in the retina, focusing mainly on the novel photic responses driven by MCs, the biochemical mechanisms triggered after light stimulation and their putative functions and implications.

Systemic C-peptide supplementation ameliorates retinal neurodegeneration by inhibiting VEGF-induced pathological events in diabetes.

Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.

Morphological changes in the brain in liver cirrhosis of alcoholic and viral etiology

Background. Hepatic encephalopathy is an actual problem of modern medicine. However, its pathogenesis and histological picture are currently insufficiently studied. Less is known about the impact of the nature of primary liver disease on pathogenesis and histological picture of hepatic encephalopathy. This determines the relevance of further morphological studies of the brain in the late stages of liver cirrhosis of various etiologies.The aim. To establish and compare the morphological changes in the brain in alcoholic liver cirrhosis and viral (hepatitis C virus (HCV)) cirrhosis.Materials and methods. The morphological study of the brain of 40 deceased in outcome of HCV-associated cirrhosis and 23 patients died in outcome of chronic alcoholism was carried out. Histological changes in various parts of the brain were studied using survey and elective stains. The immunohistochemical study of HCV NS3 and CD68 expression in different brain regions was performed in cases of HCV-infection.Results. The changes of neurons, glial cells and cerebral microvessels underlie in the basis of morphological picture of brain damage in both studied groups underlie that corresponds to the “classical” model of hepatic encephalopathy pathogenesis. At the same time, a number of morphological features were observed. The most prominent differences concerned the manifestations of the glial reaction. The productive changes of macroglial cells with the appearance of multiple Alzheimer’s astrocytes type 2 as well as spongious changes in subcortical white matter dominated in the observations of alcoholic cirrhosis. In contrast, microglia cells reaction (microgliosis) in white matter was noticed in HCV-associated cirrhosis.Conclusions. The differences in histological signs of brain in the terminal stages of liver disease of viral and alcoholic etiology are shown. They broaden current idea of morphological picture of hepatic encephalopathy, and may be used to study its pathogenesis.

Reaction of retinal macroglia to inflammatory damage

AbstractSepsis is a multi‐organ dysfunction caused by the host uncontrolled inflammatory response to an infection, mediated by proinflammatory cytokines and oxygen free radicals. LPS is unable to trespass the Blood–Brain‐Barrier (BBB), but it can act on the TLR4 (Toll‐Like Receptor 4) receptor, found in the endothelium, causing the secretion of cytokines and chemokines that lead to the activation of glial cells. On the other hand, cisplatin (CISP), also called Cis‐dichlorodiammineplatinum [II], is a heavy metal antineoplastic agent widely employed in the treatment of different forms of cancer. Neurotoxicity has been reported as some of the adverse effects resulting from CISP administration. CISP alters intracellular signal transduction pathways that, in the end, provoke apoptosis through different mechanisms such as the generation of reactive oxygen species, DNA alterations, mitochondrial and/or endoplasmic reticulum dysfunction, and the production of proinflammatory cytokines and chemokines. Both sepsis and CISP cause a systemic damage that can result in brain impairments affecting different areas of the Central Nervous System (CNS), but to our knowledge, the effects of the systemic damage over the retinal cell populations, and specially on macroglial cells, have not been studied in detail. We employed two animal models using male Wistar rats: (1) a model of sepsis based on the systemic administration of bacterial lipopolysaccharide (LPS, 10 mg/kg, i.p.), or its vehicle; to induce a systemic inflammatory response; (2) a model of cisplatin based on the administration of a daily single dose of cisplatin (CISP, 5 mg/kg, i.p.) or its vehicle for 5 consecutive days until sacrifice in order to induce neurotoxicity. Animals were transcardiacally perfused, eyes were extracted, and retinal sections were used for immunohistochemical assays, where Brn3a and GFAP were employed to stain retinal ganglion cells and macroglial cells respectively. LPS induced a reduction in the number of Brn3a+ cells in the whole retina, neurodegenerative effects being more critical in the nasal inferior retina; and provoked a generalized increase in GFAP expression, specially in the nasal and central regions of the retina. Preliminary results indicate that the CISP administration leads to a reduction in the total number of Brn3a+ cells in the retina; and CISP may induce a generalized reduction of GFAP+ cells mainly within the nasal and temporal inferior retina. Taken together, we have provided evidence of retinal damage following the systemic inflammatory processes caused by LPS and CISP administration, which affects macroglial cells, with an increased or decreased GFAP expression depending on the activation pathway of the neuroinflammatory response. This dysregulation of the macroglial response could lead to a significant diminishment of the retinal ganglion cells of the retina, causing an irreversible loss of vision.Supported by Complutense University of Madrid, UCM Research Group: 951579.Keywords: LPS‐induced sepsis, cisplatin, ganglionar and macroglial retinal cells, inflammation.

Macroglial activation over time in an experimental glaucoma model

AbstractGlaucoma is a neurodegenerative retinal disease characterized by irreversible loss of retinal ganglion cells (RGCs), leading to visual loss. Increased intraocular pressure (IOP) is the only risk factor for which treatment is available. However, other factors such as glial activation and dysfunction can also induce RGC death in glaucoma.Astrocytes and Müller glia (macroglial cells) play important roles in neuronal activity by providing physical and metabolic support to neurons. In glaucoma, when tissue damage occurs, macroglial cells undergo reactive gliosis, to defend the nerve tissue against damage and attempt to maintain its homeostasis. However, alterations in the function of these cells could cause damage and even neuronal death. In reactive macrogliosis, cells undergo complex biochemical and functional remodelling and exhibit morphological changes characterized by cell body thickening, increased number and length of cell processes, increased cell number and up‐regulation of cytoskeletal components such as gliofibrillary acidic protein (GFAP), which is considered one of the main markers of this process and may act as antigen‐presenting cells by expressing major histocompatibility complex class II (MHC‐II). Reactivation of macroglia can initially be beneficial, as it increases their metabolic activity, increases the expression of antioxidant defence, and restores ion, water and neurotransmitter balance. However, if macroglia become chronic, it is detrimental by directly or indirectly damaging the tissue and preventing its repair. Astrocytes may act in concert with microglia during the inflammatory process, so inflammatory mediators produced by astrocytes can chronically activate microglial cells, contributing to neuronal death, and similarly, inflammatory mediators released by microglia can chronically activate astrocytes.One of the most widely used models to elevate IOP is the unilateral laser‐induced ocular hypertension (OHT) model. Studies in this model have analysed the temporal pattern of RGC death and microglial cell activation at time points: 1, 3, 5, 8 and 15 days. Molecular changes in proinflammatory and anti‐inflammatory cytokines involved in the neuroinflammatory process, released by glial cells (microglia, astrocytes, and Müller cells) at the same time points mentioned above, have also been identified.Given the close relationship between the activation of macroglia and microglia and their involvement in the death of RGCs in this pathology, the present work analysed how the activation of retinal macroglial cells occurred at different time points (1, 3, 5, 8 and 15 days) after the induction of OHT in an experimental model of laser‐induced OHT in mice. For this purpose, we have analysed the GFAP‐labelled retinal area, the intensity of GFAP labelling, and the expression of MHC‐II in both OHT eyes and normotensive contralateral eyes compared to naïve eyes.In this study, in both OHT and contralateral eyes, macroglial cells showed morphological changes as well as in GFAP and MHC‐II expression at all time points analysed, being slightly more intense at 3–5 days, coinciding with the pattern of microglial activation previously studied, demonstrating this fact, a bilateral inflammatory process maintained over time in which microglia, astrocytes and Müller glia are involved.

Administration of dutasteride in animal models of retinitis pigmentosa

AbstractPurpose: Retinitis pigmentosa (RP) describes a large group of hereditary retinopathies. It mainly affects the rods. However, once the rods have degenerated, the cones, also die, leading to complete blindness. Although, the mutations that cause RP have been identified in different genes, the mechanisms that cause the death of photoreceptor cells are still unknown. Gonadal hormones may play a protective role in RP. Our research group has shown that oral administration of progesterone to RP mice significantly conserves the number of photoreceptors. 5α‐reductase inhibitors (such as dutasteride (DUT)) are approved treatments for androgenic alopecia. Modulation or inhibition of 5α‐reductase activity increases the levels of the neuroprotective steroid progesterone and 17β‐estradiol. The purpose of this study was to administer DUT to RP mice. This may be another form of increasing progesterone levels in the retina and therefore, may be neuroprotective.Methods: Animals were treated in accordance with the ARVO statement for the use of animals. rds mice were treated intraperitoneally with DUT and euthanized on day 21. Haematoxylin–eosin, cell death, and rod and cone immunohistochemistry were performed to determine if DUT was able to delay photoreceptor death. Immunohistochemical techniques were used to study changes in retinal microglia (Iba‐1) and macroglial cells. Neuronal nitric oxide synthase (nNOS) was also quantified.Results: DUT increased photoreceptor survival in rds mice. An increase in iba‐1 positive cells was found in rds retinas and DUT normalized the number, migration and the length and number of branches in these cells. DUT decreased GFAP (glial fibrillar acid protein) expression. We have also observed a slightly increase in nNOS expression in rds mice. DUT administration was also able to decrease nNOS expression.Conclusions: DUT may be used to improve cell survival, to ameliorate retinal micro and macroglial activation as well as alterations in nNOS in RP.

Role of Müller cells in cone mosaic rearrangement and retinal remodelling in photoreceptor degenerations

AbstractPhotoreceptor degenerative diseases (e.g., retinitis pigmentosa or age‐related macular degeneration) are currently one of the leading causes of irreversible blindness in the world. Although their pathophysiological mechanisms may vary, they all share some features such as initial photoreceptor loss associated with glial activation and subsequent retinal remodelling. It is well known that glial activation is an early feature of photoreceptor degenerations that is involved in retinal remodelling, which, in turn, is thought to be the cause of the secondary death of the retinal ganglion cells. The mammalian retina contains three types of glial cells. Two types of macroglial cells: astrocytes and Müller cells, and microglial cells. Of them, Müller cells appear to be involved in the reorganization of the photoreceptor mosaic in empty rings of photoreceptor degeneration in the early stages of photoreceptor loss. Müller cells are the principal glial cells in the retina with a wide range of physiological and retinal structural support functions. The basic morphological scheme of Müller cells consists of a soma located in the inner nuclear layer from which an inner and an outer trunk emerge. The inner trunk contributes to the formation of the inner limiting membrane, and the outer trunk divides into complex rootlets spanning photoreceptor nuclei and finally forming the outer limiting membrane of the retina. In the early stages of photoreceptor degeneration, macroglial cell reactivity, namely increased expression of glial fibrillary acidic protein (GFAP) and Müller cells hypertrophy, leads to the formation of a glial scar or glial seal by hypertrophic Müller cell processes in the outer retina that may be related to the formation of empty rings of photoreceptor degeneration. Interestingly, Müller cells extend their external processes horizontally and radially within the rings of photoreceptor degeneration, filling the empty space left by dead photoreceptors. These rings become larger as photoreceptor loss increases, which suggests that they are the result of photoreceptor loss. As rings increase in size and, eventually, merge, the increased expression in the processes of Müller cells continues in the periphery of the rings. Perhaps in an attempt to complete the glial seal to isolate the remnant of the neural retina from the retinal pigment epithelium and choroid which, in turn, may represent a physical barrier to axonal regrowth and migration and therefore may impede the success of therapies aimed at replacing or substituting photoreceptors. Indeed, as photoreceptor loss progresses, the glial sea is more evident, covering large areas of the retina. Although the exact function of the glial seal is not known at this stage, it is thought that it may have a mechanical function in conferring some stability to the retina during the course of degeneration, but also a protective function, since in the later stages of retinal remodelling, focal gaps in the glial seal may allow the migration of retinal pigment epithelium cells to the inner retina, triggering a series of progressive events that eventually cause retinal ganglion cell death.

Different behaviour of Müller cells according to their location in the retina: Possible relation with the differential susceptibility of retinal ganglion cells in glaucoma

AbstractPurpose: Glaucoma is a neurodegenerative disease and the leading cause of irreversible blindness in the world. Increased intraocular pressure (IOP) is considered a major risk factor. When visual problems are first detected in patients with glaucoma, half of the retinal ganglion cells (RGCs) have already died, principally in the peripheral retina. Müller glia (MG), which are the main macroglial cells in the retina, play prominent physiological and pathological roles. The change of MG to a reactive phenotype initiates signalling cascades that may serve a neuroprotective role, but may also promote damaging effects on RGCs. Our main hypothesis is that MG exert an important role in RGCs death in glaucoma, being the MG in the periphery the most sensitive to intraocular pressure, promoting the death of RGCs.Methods: Survival of RGCs was analysed by immunocytochemistry in porcine primary co‐cultures of RGCs and MG from central and peripheral retina under control and elevated hydrostatic pressure (EHP) conditions. The conditioned media (CM) from MG primary cultures from central and peripheral retina were collected and analysed by mass spectrometry to identify and quantify proteins in all experimental conditions.Results: We demonstrated that in control condition MG secret neuroprotective factors, and in EHP MG contribute to RGC death. MG from central retina are less sensitive to EHP since RGCs survival further decrease in co‐culture with MG from peripheral retina. Moreover, stretch‐activated nonselective TRPV4 cation channels present in MG are differently activated by the pressure depending of their location within the retina. Proteomic analysis from CM of MG revealed differential protein expression levels, being more pronounced in the CM from MG from peripheral retina under EHP. The proteins identified are mainly associated to apoptosis, oxidative stress, inflammation and angiogenesis.Conclusions: The different sensitivity of the RGCs to IOP in glaucoma could be related with the different activation of neighbouring MG.

Müller‐retinal ganglion cell interactions

AbstractGlaucoma is a neurodegenerative disease and the leading cause of irreversible blindness in the world, whose main risk factor is increased intraocular pressure. When visual problems are first detected in patients with glaucoma, already half of the retinal ganglion cells (RGCs), the neurons that send visual information from the eye to the brain, have died principally in the peripheral retina. The timeframe of cell death is prolonged, lasting even decades after an initial diagnosis. Müller glia (MG) are the main macroglial cell in the retina, play prominent roles regulating normal and pathological physiology and react to neuronal injury in glaucoma. The change to a reactive phenotype of MG initiates signalling cascades that may serve a neuroprotective role but may also proceed to promote damaging effects on RGCs. However, the underlying mechanisms and signalling pathways that specifically promote protective versus destructive roles of reactive glial cells are mostly unclear.Our main hypothesis is that MGs exert an important role in the death of RGCs in glaucoma with the MGs in the periphery being the most sensitive to intraocular pressure, promoting the death of RGCs. Preventing MG response to elevated pressure, MG can neuroprotect RGCs in early stages of glaucoma.We have proved in vitro, that in control condition MG secret neuroprotective factors, and under elevated pressure MG are the responsible for the RGCs death, being less sensitive to the elevated hydrostatic pressure (EHP) the MG present in the central retina. Moreover, stretch‐activated nonselective TRPV4 cation channels present in MG are differently activated by the pressure depending on their location within the retina. Proteomic analysis from the conditioned media (CM) of MG revealed different protein expression levels being more pronounced in the CM from MG from peripheral retina in EHP. Proteins identified correspond mainly to functions such as: apoptosis, oxidative stress, inflammation and angiogenesis.We conclude that the different sensibility of the RGCs to pressure in glaucoma could be related with the different activation of the neighbours MG.Supported by ELKARTEK (KK‐2019/00086), MINECO‐Retos (PID2019‐111139RB‐I00) and PIBA (2020_1_0026).

Understanding the role of microglia in the onset of photoreceptor degeneration

AbstractPhotoreceptor degenerative diseases are currently the leading cause of irreversible blindness in developed countries and a major cause of irreversible blindness in the world. The most common forms of these diseases are age‐related macular degeneration and retinitis pigmentosa, both diseases cause a profound visual impairment due to photoreceptor loss. In the early stages of photoreceptor degeneration, glial cells have a major role to play, presumably leading to extensive retinal remodelling. The mammalian retina contains three types of glial cells. Two types of macroglial cells: astrocytes and Müller cells, and microglial cells. Microglial cells are the major resident immune cells in the retina and, although these cells and neuroinflammation have been implicated in the pathology of many neurodegenerative diseases, their role and function in photoreceptor degenerations are not yet fully understood. During retinal degeneration, microglial cells change their morphology, become activated and travel from the inner to the outer layers of the retina to phagocytose the dying photoreceptors, while astrocytes and Müller cells become hypertrophic, and overexpress glial fibrillary acidic protein (GFAP) filling the space left by dead photoreceptors to form a glial seal. It is known that activated microglial cells can be either neuroprotective or neurodestructive during photoreceptor degenerations, and therefore it is not yet clear whether activated microglial cells may increase or decrease photoreceptor loss. Microglial cell activation, migration and proliferation occur simultaneously with the onset of photoreceptor degeneration, prior to the loss of the vast majority of photoreceptors. During photoreceptor loss, microglial cells reach the outer nuclear and outer segment layers. These events suggest that photoreceptor death is the trigger for the activation and migration of microglia to the outer retinal layers, where they phagocytose the dying photoreceptors and remove cellular debris. By carrying out these functions, microglial cells in the retina may affect photoreceptor survival or death, eventually phagocytizing stressed but still viable photoreceptors. Interestingly, inhibition of microglial cell activation and migration during photoreceptor degeneration results in improved cone outer segment morphology and increased photoreceptor survival, which suggest that the role of activated microglial cells may be more neurodestructive than neuroprotective and that treatments with anti‐inflammatory drugs that modulate microglial reactivity may be considered in the early stages of photoreceptor degenerations.