Search

Your search keyword '"Ruiz‐Lopez, Ana M."' showing total 15 results
Start Over Author "Ruiz‐Lopez, Ana M."
15 results on '"Ruiz‐Lopez, Ana M."'

6. The synthetic progestin norgestrel acts to increase LIF levels in the rd10 mouse model of retinitis pigmentosa

Author

Byrne, Ashleigh M., Roche, Sarah L., Ruiz-Lopez, Ana M., Jackson, Alice C. Wyse, and Cotter, Thomas G.

Subjects
Cell Survival, Blotting, Western, Norgestrel, Real-Time Polymerase Chain Reaction, Transfection, Contraceptives, Oral, Synthetic, Leukemia Inhibitory Factor, Diet, Mice, Inbred C57BL, Disease Models, Animal, Mice, Gene Expression Regulation, Animals, sense organs, RNA, Messenger, RNA, Small Interfering, Fluorescent Antibody Technique, Indirect, Cells, Cultured, Retinitis Pigmentosa, Research Article, Photoreceptor Cells, Vertebrate
Abstract

Purpose: Retinal degenerative conditions affect thousands of people worldwide. Retinitis pigmentosa (RP) is among the most common, but it is currently incurable. It is characterized by the progressive death of photoreceptor cells, eventually leading to blindness. Neurotrophic factors play an important role in such retinopathies, and much research has been performed on their use as treatments. Our group previously demonstrated the ability of the synthetic progestin norgestrel to rescue photoreceptors from cell death, the mechanism of which is believed to include upregulation of the neurotrophic factor basic fibroblast growth factor (bFGF). The objective of the present study was to investigate whether the protection provided by norgestrel is likely to be mediated by other neurotrophins. Methods: The 661W photoreceptor cells and retinal explants from P30 to P40 wild-type (wt) C57BL/6 mice were treated with norgestrel over time. Homozygous rd10/rd10 mice that mimic the human form of RP were fed either a control or a norgestrel-containing diet. Changes in neurotrophic factor expression in response to norgestrel were detected with real-time PCR, western blotting, or immunofluorescence staining. Using specific siRNA, leukemia inhibitory factor (Lif) expression was knocked down in 661W photoreceptor cells that were stressed by serum starvation. Cells were treated with norgestrel followed by measurement of cell viability with (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Results: LIF, a potent neuroprotective cytokine, was found to be upregulated in response to norgestrel in vitro and in vivo. Upregulation of LIF in degenerating rd10 retinas coincided with preservation of the photoreceptor layer. We also found LIF was necessary for the norgestrel-mediated rescue of stressed photoreceptor cells from cell death in vitro. Conclusions: LIF was upregulated in response to norgestrel in all models studied and is necessary for the protective effects of norgestrel in vitro. The increase in LIF expression in rd10 mice undergoing retinal degeneration was concurrent with rescue of the photoreceptor cell layer. These results highlight the ability of norgestrel to induce prosurvival molecules in the compromised retina, underlining norgestrel’s potential as a viable drug for treatment of RP.

Published
2016

10. Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

Author

Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Roche, Sarah L., Wyse-Jackson, Alice C., Gómez-Vicente, Violeta, Lax, Pedro, Ruiz-Lopez, Ana M., Byrne, Ashleigh M., Cuenca, Nicolás, Cotter, Thomas G., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Roche, Sarah L., Wyse-Jackson, Alice C., Gómez-Vicente, Violeta, Lax, Pedro, Ruiz-Lopez, Ana M., Byrne, Ashleigh M., Cuenca, Nicolás, and Cotter, Thomas G.

Abstract

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising tre

Published
2016

15. Cell Death Analysis in Retinal Cultures.

Author

Roche SL, Ruiz-Lopez AM, and Cotter TG

Subjects
Animals, Apoptosis, Biomarkers, Cell Line, Cell Survival, Immunohistochemistry methods, Mice, Microscopy, Fluorescence, Photoreceptor Cells, Vertebrate metabolism, Cell Death, Organ Culture Techniques, Retina metabolism
Abstract

Evaluating cell death is essential when investigating neurodegeneration and neuroprotection in the retina. Cell death assays provide us with a means to identify and quantify dying cells in a population. Terminal dUTP nick end-labeling (TUNEL) is one method used for the identification of dying cells. This technique is based upon the enzymatic incorporation of fluorescently tagged DNA base pairs to fragmented DNA. In this chapter, we describe two different techniques employing TUNEL. The first method uses TUNEL to analyze cell death in cultured retinal explants by fluorescence microscopy. The second technique describes a method for measuring cell death in a retinal cell line by flow cytometry.

Published
2019
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results