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Microscopy and Microanalysis : the... Oct 2018Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level...
Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as "living light guides" as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.
Topics: Animals; Cytoplasm; Ependymoglial Cells; Eye; Immunohistochemistry; Lens, Crystalline; Light; Microscopy, Immunoelectron; Neuroglia; Optical Imaging; Photoreceptor Cells; Rats; Rats, Sprague-Dawley; Retina; Retinal Rod Photoreceptor Cells; alpha-Crystallin A Chain; alpha-Crystallins
PubMed: 30253817
DOI: 10.1017/S1431927618015118 -
Molecular Vision 2020To analyze whether activation of endogenous wingless (Wnt)/β-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following...
PURPOSE
To analyze whether activation of endogenous wingless (Wnt)/β-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage.
METHODS
Transgenic mice with a tamoxifen-dependent β-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal β-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed.
RESULTS
Following NMDA injection of wild-type mice, a statistically significant increase in retinal β-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific β-catenin deficiency. Furthermore, in mice with a β-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a β-catenin deficiency in Müller cells.
CONCLUSIONS
Endogenous Wnt/β-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.
Topics: Animals; Apoptosis; Axons; Ependymoglial Cells; In Situ Nick-End Labeling; Leukemia Inhibitory Factor; Mice; Mice, Transgenic; N-Methylaspartate; Optic Nerve; Retina; Retinal Ganglion Cells; Tamoxifen; Wnt Signaling Pathway; beta Catenin
PubMed: 32180679
DOI: No ID Found -
Investigative Ophthalmology & Visual... Jul 2023Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of...
Phosphorylation of αB-Crystallin Involves Interleukin-1β-Mediated Intracellular Retention in Retinal Müller Cells: A New Mechanism Underlying Fibrovascular Membrane Formation.
PURPOSE
Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of the key elements. The phosphorylation of αB-crystallin/CRYAB modulates its molecular dynamics and chaperone activity, and attenuates αB-crystallin secretion via exosomes. In this study, we investigated the effect of phosphorylated αB-crystallin in retinal Müller cells on diabetic mimicking conditions, including interleukin (IL)-1β stimuli.
METHODS
Human retinal Müller cells (MIO-M1) were used to examine gene and protein expressions with real-time quantitative PCR, enzyme linked immunosorbent assay (ELISA), and immunoblot analyses. Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling staining. Retinal tissues isolated from the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic animal model with obesity, and fibrovascular membranes from patients with PDR were examined by double-staining immunofluorescence.
RESULTS
CRYAB mRNA was downregulated in MIO-M1 cells with the addition of 10 ng/mL IL-1β; however, intracellular αB-crystallin protein levels were maintained. The αB-crystallin serine 59 (Ser59) residue was phosphorylated with IL-1β application in MIO-M1 cells. Cell apoptosis in MIO-M1 cells was induced by CRYAB knockdown. Immunoreactivity for Ser59-phosphorylated αB-crystallin and glial fibrillary acidic protein was colocalized in glial cells of SDT fatty rats and fibrovascular membranes.
CONCLUSIONS
The Ser59 phosphorylation of αB-crystallin was modulated by IL-1β in Müller cells under diabetic mimicking inflammatory conditions, suggesting that αB-crystallin contributes to the pathogenesis of PDR through an anti-apoptotic effect.
Topics: Humans; Rats; Animals; Phosphorylation; Ependymoglial Cells; Interleukin-1beta; alpha-Crystallin B Chain; Apoptosis; Diabetic Retinopathy
PubMed: 37459063
DOI: 10.1167/iovs.64.10.20 -
Biomolecules Apr 2024Augmenting the natural melanocortin pathway in mouse eyes with uveitis or diabetes protects the retinas from degeneration. The retinal cells are protected from oxidative...
Augmenting the natural melanocortin pathway in mouse eyes with uveitis or diabetes protects the retinas from degeneration. The retinal cells are protected from oxidative and apoptotic signals of death. Therefore, we investigated the effects of a therapeutic application of the melanocortin alpha-melanocyte-stimulating hormone (α-MSH) on an ischemia and reperfusion (I/R) model of retinal degenerative disease. Eyes were subjected to an I/R procedure and were treated with α-MSH. Retinal sections were histopathologically scored. Also, the retinal sections were immunostained for viable ganglion cells, activated Muller cells, microglial cells, and apoptosis. The I/R caused retinal deformation and ganglion cell loss that was significantly reduced in I/R eyes treated with α-MSH. While α-MSH treatment marginally reduced the number of GFAP-positive Muller cells, it significantly suppressed the density of Iba1-positive microglial cells in the I/R retinas. Within one hour after I/R, there was apoptosis in the ganglion cell layer, and by 48 h, there was apoptosis in all layers of the neuroretina. The α-MSH treatment significantly reduced and delayed the onset of apoptosis in the retinas of I/R eyes. The results demonstrate that therapeutically augmenting the melanocortin pathways preserves retinal structure and cell survival in eyes with progressive neuroretinal degenerative disease.
Topics: Animals; alpha-MSH; Reperfusion Injury; Mice; Apoptosis; Retina; Homeostasis; Retinal Ganglion Cells; Mice, Inbred C57BL; Microglia; Male; Ependymoglial Cells; Disease Models, Animal; Retinal Degeneration
PubMed: 38785932
DOI: 10.3390/biom14050525 -
Journal of Cellular and Molecular... Jan 2021We investigated how Src-homology 2-domain phosphatase-1 (SHP-1) regulates the inflammatory response in endotoxin-induced uveitis (EIU), and the signalling pathways...
We investigated how Src-homology 2-domain phosphatase-1 (SHP-1) regulates the inflammatory response in endotoxin-induced uveitis (EIU), and the signalling pathways involved. One week after intravitreal injection of short hairpin RNA targeting SHP-1 or SHP-1 overexpression lentivirus in rats, we induced ocular inflammation with an intravitreal injection of lipopolysaccharide (LPS). We then assessed the extent of inflammation and performed full-field electroretinography. The concentrations and retinal expression of various inflammatory mediators were examined with enzyme-linked immunosorbent assays and Western blotting, respectively. SHP-1 overexpression and knockdown were induced in Müller cells to study the role of SHP-1 in the LPS-induced inflammatory response in vitro. Retinal SHP-1 expression was up-regulated by LPS. SHP-1 knockdown exacerbated LPS-induced retinal dysfunction and increased the levels of proinflammatory mediators in the retina, which was abrogated by a c-Jun N-terminal kinase (JNK) inhibitor (SP600125). SHP-1 overexpression had the opposite effects. In Müller cells, the LPS-induced inflammatory response was enhanced by SHP-1 knockdown and suppressed by SHP-1 overexpression. SHP-1 negatively regulated the activation of the transforming growth factor-β-activated kinase-1 (TAK1)/JNK pathway, but not the nuclear factor-κB pathway. These results indicate that SHP-1 represses EIU, at least in part, by inhibiting the TAK1/JNK pathway and suggest that SHP-1 is a potential therapeutic target for uveitis.
Topics: Animals; Anterior Chamber; Aqueous Humor; Cytokines; Down-Regulation; Endotoxins; Ependymoglial Cells; Inflammation; JNK Mitogen-Activated Protein Kinases; Lipopolysaccharides; MAP Kinase Kinase Kinases; Male; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Rats, Sprague-Dawley; Retina; Up-Regulation; Uveitis; Vitreous Body; Rats
PubMed: 33207073
DOI: 10.1111/jcmm.15888 -
Investigative Ophthalmology & Visual... Mar 2017Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal...
PURPOSE
Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Müller cell extension into the subretinal space.
METHODS
Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and vimentin (Müller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Müller cells.
RESULTS
Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry.
CONCLUSIONS
In eyes with GA, subretinal Müller cell membranes present in areas of RPE atrophy may be a Müller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy.
Topics: Aged; Aged, 80 and over; Astrocytes; Cell Membrane; Ependymoglial Cells; Female; Geographic Atrophy; Humans; Immunohistochemistry; Male; Microscopy, Confocal; Microscopy, Electron, Transmission; Retina; Vimentin
PubMed: 28249091
DOI: 10.1167/iovs.16-21229 -
Journal of Extracellular Vesicles Sep 2022Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through...
Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo. We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro. Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.
Topics: Animals; Ependymoglial Cells; Extracellular Vesicles; Mice; Neuroglia; Neurons; Retina
PubMed: 36043482
DOI: 10.1002/jev2.12254 -
International Journal of Molecular... Feb 2021Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons,...
Proliferative diabetic retinopathy (PDR), a major complication of diabetes mellitus, results from an inflammation-sustained interplay among endothelial cells, neurons, and glia. Even though anti-vascular endothelial growth factor (VEGF) interventions represent the therapeutic option for PDR, they are only partially efficacious. In PDR, Müller cells undergo reactive gliosis, produce inflammatory cytokines/chemokines, and contribute to scar formation and retinal neovascularization. However, the impact of anti-VEGF interventions on Müller cell activation has not been fully elucidated. Here, we show that treatment of MIO-M1 Müller cells with vitreous obtained from PDR patients stimulates cell proliferation and motility, and activates various intracellular signaling pathways. This leads to cytokine/chemokine upregulation, a response that was not mimicked by treatment with recombinant VEGF nor inhibited by the anti-VEGF drug ranibizumab. In contrast, fibroblast growth factor-2 (FGF2) induced a significant overexpression of various cytokines/chemokines in MIO-M1 cells. In addition, the FGF receptor tyrosine kinase inhibitor BGJ398, the pan-FGF trap NSC12, the heparin-binding protein antagonist N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe Boc2, and the anti-inflammatory hydrocortisone all inhibited Müller cell activation mediated by PDR vitreous. These findings point to a role for various modulators beside VEGF in Müller cell activation and pave the way to the search for novel therapeutic strategies in PDR.
Topics: Aged; Cell Proliferation; Cells, Cultured; Cholesterol; Diabetic Retinopathy; Ependymoglial Cells; Female; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Hydrocortisone; Inflammation Mediators; Male; Middle Aged; Phenylurea Compounds; Pyrimidines; Ranibizumab; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A; Vitrectomy
PubMed: 33671690
DOI: 10.3390/ijms22042179 -
International Ophthalmology Dec 2023This study investigated the protective effect of probucol on Müller cells exposed to high glucose conditions and examined potential mechanisms of action.
PURPOSE
This study investigated the protective effect of probucol on Müller cells exposed to high glucose conditions and examined potential mechanisms of action.
METHODS
Primary human retinal Müller cells were incubated with high glucose (HG, 35 mM) in the present or absence of different concentrations of probucol for 24 h. Cell viability was determined using the CCK-8 method. Mitochondrial membrane potential (MMP) was measured using JC-1 staining and cell cycle by flow cytometry. The expression of nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit, and p62 was quantified using quantitative polymerase chain reaction and western blot.
RESULTS
We found that HG inhibited cell proliferation, arrested cell cycle, and increased MMP in human Müller cells. Probucol activated the Nrf2/p62 pathway and upregulated the anti-apoptotic protein, Bcl2, and attenuated HG-mediated damage in Müller cells.
CONCLUSIONS
Our results suggest that probucol may protect Müller cells from HG-induced damage through enhancing the Nrf2/p62 signaling pathway.
Topics: Humans; Ependymoglial Cells; Glucose; NF-E2-Related Factor 2; Probucol; Signal Transduction
PubMed: 37688651
DOI: 10.1007/s10792-023-02859-z -
Clinics (Sao Paulo, Brazil) 2023To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR).
OBJECTIVES
To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR).
METHOD
The expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1β, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1β, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1β and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA).
RESULTS
Compared with the surrounding normal tissues, the expression of caspase-1, IL-1β, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05).
CONCLUSIONS
MCP can promote the development of PVR lesions.
Topics: Humans; Vitreoretinopathy, Proliferative; Interleukin-18; Pyroptosis; Ependymoglial Cells; Cytokines; RNA, Messenger; Caspases
PubMed: 37418795
DOI: 10.1016/j.clinsp.2023.100241