Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses

The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adu...

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Autores principales: Meneses, Constanza Stefania, Müller, Heine Yacob, Herzberg, Daniel Eduardo, Uberti, Benjamín, Bustamante, Hedie Almagro, Werner, Marianne Patricia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661433/
https://www.ncbi.nlm.nih.gov/pubmed/29085760
http://dx.doi.org/10.7717/peerj.3965
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author Meneses, Constanza Stefania
Müller, Heine Yacob
Herzberg, Daniel Eduardo
Uberti, Benjamín
Bustamante, Hedie Almagro
Werner, Marianne Patricia
author_facet Meneses, Constanza Stefania
Müller, Heine Yacob
Herzberg, Daniel Eduardo
Uberti, Benjamín
Bustamante, Hedie Almagro
Werner, Marianne Patricia
author_sort Meneses, Constanza Stefania
collection PubMed
description The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.
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spelling pubmed-56614332017-10-30 Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses Meneses, Constanza Stefania Müller, Heine Yacob Herzberg, Daniel Eduardo Uberti, Benjamín Bustamante, Hedie Almagro Werner, Marianne Patricia PeerJ Molecular Biology The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses. PeerJ Inc. 2017-10-27 /pmc/articles/PMC5661433/ /pubmed/29085760 http://dx.doi.org/10.7717/peerj.3965 Text en ©2017 Meneses et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Molecular Biology
Meneses, Constanza Stefania
Müller, Heine Yacob
Herzberg, Daniel Eduardo
Uberti, Benjamín
Bustamante, Hedie Almagro
Werner, Marianne Patricia
Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
title Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
title_full Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
title_fullStr Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
title_full_unstemmed Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
title_short Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
title_sort immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661433/
https://www.ncbi.nlm.nih.gov/pubmed/29085760
http://dx.doi.org/10.7717/peerj.3965
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