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Article ; Online: Protective Effect of NO

Vaglienti, María V / Subirada, Paula V / Joray, Mariana B / Bonacci, Gustavo / Sánchez, María C

2023 Volume 12, Issue 3

Abstract: Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor ...

Abstract	Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α
MeSH term(s)	Humans ; Nitrogen Dioxide/metabolism ; Nitrogen Dioxide/pharmacology ; Vascular Endothelial Growth Factor A/metabolism ; Ependymoglial Cells/metabolism ; Gliosis/metabolism ; Oxidative Stress ; Hypoxia/metabolism ; Inflammation/metabolism ; RNA, Messenger/metabolism
Chemical Substances	Nitrogen Dioxide (S7G510RUBH) ; Vascular Endothelial Growth Factor A ; RNA, Messenger
Language	English
Publishing date	2023-02-02
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2661518-6
ISSN	2073-4409 ; 2073-4409
ISSN (online)	2073-4409
ISSN	2073-4409
DOI	10.3390/cells12030494
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Quantification of reactive oxygen species using 2′,7′-dichlorofluorescein diacetate probe and flow-cytometry in müller glial cells

Vaglienti, María V. / Subirada, Paula V. / Barcelona, Pablo F. / Bonacci, Gustavo / Sanchez, María C.

Journal of visualized experiments. 2022 May 13, , no. 183

2022

Abstract: The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and ... ...

Abstract	The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, β-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.
Keywords	DNA ; antioxidant activity ; cell death ; computer software ; diabetic retinopathy ; flow cytometry ; fluorescence ; homeostasis ; inflammation ; models ; oxidative stress ; pathogenesis ; polyphenols ; premature birth ; protective effect ; reactive oxygen species ; retina
Language	English
Dates of publication	2022-0513
Size	p. e63337.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/63337
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Quantification of Reactive Oxygen Species Using 2',7'-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells.

Vaglienti, María V / Subirada, Paula V / Barcelona, Pablo F / Bonacci, Gustavo / Sanchez, María C

Journal of visualized experiments : JoVE

2022 , Issue 183

Abstract	The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, β-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.
MeSH term(s)	Antioxidants/metabolism ; Antioxidants/pharmacology ; Ependymoglial Cells ; Flow Cytometry ; Fluoresceins ; Humans ; Infant, Newborn ; Kelch-Like ECH-Associated Protein 1/metabolism ; NF-E2-Related Factor 2/metabolism ; Reactive Oxygen Species/metabolism
Chemical Substances	Antioxidants ; Fluoresceins ; Kelch-Like ECH-Associated Protein 1 ; NF-E2-Related Factor 2 ; Reactive Oxygen Species ; diacetyldichlorofluorescein (2044-85-1)
Language	English
Publishing date	2022-05-13
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/63337
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Quantification of vascular parameters in whole mount retinas of mice with non-proliferative and proliferative retinopathies

Subirada, Paula V. / Paz, María C. / Vaglienti, María V. / Luna, José D. / Barcelona, Pablo F. / Sánchez, María C.

Journal of visualized experiments. 2022 Mar. 12, , no. 181

2022

Abstract: Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis and a progressive change in vascular function and structure. Although the onset of the retinopathies ... ...

Abstract	Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.
Keywords	angiogenesis ; computer software ; death ; endothelium ; neurodegenerative diseases ; neurons ; retina ; retinal diseases ; visual perception ; Fiji
Language	English
Dates of publication	2022-0312
Size	p. e63126.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/63126
Database	NAL-Catalogue (AGRICOLA)

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Article: Rapamycin and Resveratrol Modulate the Gliotic and Pro-Angiogenic Response in Müller Glial Cells Under Hypoxia.

Subirada, Paula V / Vaglienti, María V / Joray, Mariana B / Paz, María C / Barcelona, Pablo F / Sánchez, María C

Frontiers in cell and developmental biology

2022 Volume 10, Page(s) 855178

Abstract: Hypoxia and hypoxia-reoxygenation are frequently developed through the course of many retinal diseases of different etiologies. Müller glial cells (MGCs), together with microglia and astrocytes, participate firstly in response to the injury and later in ... ...

Abstract	Hypoxia and hypoxia-reoxygenation are frequently developed through the course of many retinal diseases of different etiologies. Müller glial cells (MGCs), together with microglia and astrocytes, participate firstly in response to the injury and later in the repair of tissue damage. New pharmacological strategies tend to modulate MGCs ability to induce angiogenesis and gliosis in order to accelerate the recovery stage. In this article, we investigated the variation in autophagy flux under hypoxia during 4 h, employing both gas culture chamber (1% O
Language	English
Publishing date	2022-03-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2022.855178
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies.

Subirada, Paula V / Paz, María C / Vaglienti, María V / Luna, José D / Barcelona, Pablo F / Sánchez, María C

Journal of visualized experiments : JoVE

2022 , Issue 181

Abstract	Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.
MeSH term(s)	Animals ; Eye Diseases ; Mice ; Neovascularization, Pathologic ; Retina ; Retinal Diseases ; Retinal Vessels
Language	English
Publishing date	2022-03-12
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/63126
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Metabolic Syndrome Triggered by Fructose Diet Impairs Neuronal Function and Vascular Integrity in ApoE-KO Mouse Retinas: Implications of Autophagy Deficient Activation.

Paz, María C / Barcelona, Pablo F / Subirada, Paula V / Ridano, Magali E / Chiabrando, Gustavo A / Castro, Claudia / Sánchez, María C

Frontiers in cell and developmental biology

2020 Volume 8, Page(s) 573987

Abstract: Metabolic syndrome is a disorder characterized by a constellation of clinical findings such as elevated blood glucose, hyperinsulinemia, dyslipidemia, hypertension, and obesity. A positive correlation has been found between metabolic syndrome or its ... ...

Abstract	Metabolic syndrome is a disorder characterized by a constellation of clinical findings such as elevated blood glucose, hyperinsulinemia, dyslipidemia, hypertension, and obesity. A positive correlation has been found between metabolic syndrome or its components and retinopathy, mainly at microvascular level, in patients without a history of diabetes. Here, we extend the investigations beyond the vascular component analyzing functional changes as well as neuronal and glial response in retinas of Apolipoprotein E knockout (ApoE-KO) mice fed with 10% w/v fructose diet. Given that autophagy dysfunction is implicated in retinal diseases related to hyperglycemia and dyslipidemia, the activation of this pathway was also analyzed. Two months of fructose intake triggered metabolic derangements in ApoE-KO mice characterized by dyslipidemia, hyperglycemia and hyperinsulinemia. An increased number of TUNEL positive cells, in addition to the ganglion cell layer, was observed in the inner nuclear layer in retina. Vascular permeability, evidenced by albumin-Evans blue leakage and extravasation of albumin was also detected. Furthermore, a significant decrease of the glial fibrillary acidic protein expression was confirmed by Western blot analysis. Absence of both Müller cell gliosis and pro-angiogenic response was also demonstrated. Finally, retinas of ApoE-KO FD mice showed defective autophagy activation as judged by LC3B mRNA and p62 protein levels correlating with the increased cell death. These results demonstrated that FD induced in ApoE-KO mice biochemical alterations compatible with metabolic syndrome associated with neuronal impairment and mild vascular alterations in the retina.
Language	English
Publishing date	2020-10-08
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2020.573987
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Effect of Autophagy Modulators on Vascular, Glial, and Neuronal Alterations in the Oxygen-Induced Retinopathy Mouse Model.

Subirada, Paula V / Paz, María C / Ridano, Magali E / Lorenc, Valeria E / Fader, Claudio M / Chiabrando, Gustavo A / Sánchez, María C

Frontiers in cellular neuroscience

2019 Volume 13, Page(s) 279

Abstract: Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, ... ...

Abstract	Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.
Language	English
Publishing date	2019-06-26
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2452963-1
ISSN	1662-5102
ISSN	1662-5102
DOI	10.3389/fncel.2019.00279
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: A journey into the retina: Müller glia commanding survival and death.

Subirada, Paula V / Paz, María C / Ridano, Magali E / Lorenc, Valeria E / Vaglienti, María V / Barcelona, Pablo F / Luna, José D / Sánchez, María C

The European journal of neuroscience

2018 Volume 47, Issue 12, Page(s) 1429–1443

Abstract: Müller glial cells (MGCs) are known to participate actively in retinal development and to contribute to homoeostasis through many intracellular mechanisms. As there are no homologous cells in other neuronal tissues, it is certain that retinal health ... ...

Abstract	Müller glial cells (MGCs) are known to participate actively in retinal development and to contribute to homoeostasis through many intracellular mechanisms. As there are no homologous cells in other neuronal tissues, it is certain that retinal health depends on MGCs. These macroglial cells are located at the centre of the columnar subunit and have a great ability to interact with neurons, astrocytes, microglia and endothelial cells in order to modulate different events. Several investigations have focused their attention on the role of MGCs in diabetic retinopathy, a progressive pathology where several insults coexist. As expected, data suggest that MGCs display different responses according to the severity of the stimulus, and therefore trigger distinct events throughout the course of the disease. Here, we describe physiological functions of MGCs and their participation in inflammation, gliosis, synthesis and secretion of trophic and antioxidant factors in the diabetic retina. We invite the reader to consider the protective/deleterious role of MGCs in the early and late stages of the disease. In the light of the results, we open up the discussion around and ask the question: Is it possible that the modulation of a single cell type could improve or even re-establish retinal function after an injury?
MeSH term(s)	Animals ; Diabetic Retinopathy/immunology ; Diabetic Retinopathy/metabolism ; Diabetic Retinopathy/physiopathology ; Ependymoglial Cells/immunology ; Ependymoglial Cells/metabolism ; Ependymoglial Cells/physiology ; Gliosis/immunology ; Gliosis/metabolism ; Gliosis/physiopathology ; Humans ; Inflammation/immunology ; Inflammation/metabolism ; Inflammation/physiopathology ; Nerve Growth Factors/immunology ; Nerve Growth Factors/metabolism ; Nerve Growth Factors/physiology ; Oxidative Stress/immunology ; Oxidative Stress/physiology
Chemical Substances	Nerve Growth Factors
Language	English
Publishing date	2018-06-08
Publishing country	France
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	645180-9
ISSN	1460-9568 ; 0953-816X
ISSN (online)	1460-9568
ISSN	0953-816X
DOI	10.1111/ejn.13965
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Galectin-1 expression imprints a neurovascular phenotype in proliferative retinopathies and delineates responses to anti-VEGF.

Ridano, Magali E / Subirada, Paula V / Paz, María C / Lorenc, Valeria E / Stupirski, Juan C / Gramajo, Ana L / Luna, José D / Croci, Diego O / Rabinovich, Gabriel A / Sánchez, María C

Oncotarget

2017 Volume 8, Issue 20, Page(s) 32505–32522

Abstract: Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As ... ...

Abstract	Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1-/-) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.
Language	English
Publishing date	2017-05-16
Publishing country	United States
Document type	Journal Article
ZDB-ID	2560162-3
ISSN	1949-2553 ; 1949-2553
ISSN (online)	1949-2553
ISSN	1949-2553
DOI	10.18632/oncotarget.17129
Database	MEDical Literature Analysis and Retrieval System OnLINE

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