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Home / Equine Research Bank / PeerJ / Immunofluorescence characterization of spinal cord dorsal ho...
PeerJ2017; 5; e3965; doi: 10.7717/peerj.3965

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

Abstract: 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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Publication Date: 2017-10-27 PubMed ID: 29085760PubMed Central: PMC5661433DOI: 10.7717/peerj.3965Google Scholar: Lookup
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Summary

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This study examines the structure and arrangement of healthy spinal microglia (a type of brain cell) and astrocyte (another type of brain cell) in horses. The researchers used specific techniques to study these cells in the spinal cord of horses, and found that their shape and size is similar to those found in rodents, but different from those seen in humans.

Study Objective and Methodology

  • The goal of the study was to understand the shape, size and arrangement of healthy spinal microglia and astrocyte cells in horses. These cells are part of the glial cell family, which recently drawn attention for their role in pain regulation.
  • The researchers studied five adult horses that showed no signs of injuries or abnormal gait, and took spinal cord segment samples after they died.
  • Using immunofluorescence labeling, the cells of interest were highlighted for further examination. A technique called immunohistochemistry was used to visualize the cells, and the researchers used specific antibodies to bind to and highlight the astrocytes and microglial cells in the horse’s spinal cord.

Findings on Microglia and Astrocyte cells

  • The researchers found that the characteristic shape usually seen in resting microglial cells was not present in horses. Instead, these cells appeared spherical and amoeba-like.
  • The primary branches of astrocytes, which are fewer in number and less complex than humans, were mainly detected using an antibody against Glial Fibrillary Acidic Protein (GFAP).
  • Horse astrocytes are similar in size and placement to rodent astrocytes. They have a smaller diameter, with primary process lengths around 28µm and thicknesses of 1.4µm.
  • The researchers found a close relationship between the end-point processes of astrocytes and the bodies of microglial cells.

Significance of the Research

  • This study provides the first detailed description of the morphology and organization of spinal glial cells in horses.
  • The researchers successfully used Iba-1 and GFAP/Cx-43 to label and observe microglia and astrocytes respectively in horse spinal cords. This provided clear images of the cell shapes and their distribution within the spinal cord in healthy horses.
  • Understanding the shape, size, and arrangement of these cells in horses could provide a foundation for further studies into the roles these cells play in health and disease, particularly in relation to pain modulation.

+Life_Science

Cite This Article

APA
Meneses CS, Müller HY, Herzberg DE, Uberti B, Bustamante HA, Werner MP. (2017). Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses. PeerJ, 5, e3965. https://doi.org/10.7717/peerj.3965

Publication

PeerJ
ISSN: 2167-8359
NlmUniqueID: 101603425
Country: United States
Language: English
Volume: 5
Pages: e3965

Researcher Affiliations

Meneses, Constanza Stefania
  • Veterinary Sciences Graduate School, Universidad Austral de Chile, Valdivia, Chile.
Müller, Heine Yacob
  • Veterinary Sciences Graduate School, Universidad Austral de Chile, Valdivia, Chile.
Herzberg, Daniel Eduardo
  • Veterinary Sciences Graduate School, Universidad Austral de Chile, Valdivia, Chile.
Uberti, Benjamín
  • Veterinary Clinical Sciences Department, Universidad Austral de Chile, Valdivia, Chile.
Bustamante, Hedie Almagro
  • Veterinary Clinical Sciences Department, Universidad Austral de Chile, Valdivia, Chile.
Werner, Marianne Patricia
  • Animal Science Department, Universidad Austral de Chile, Valdivia, Chile.

Conflict of Interest Statement

The authors declare there are no competing interests.

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Citations

This article has been cited 3 times.
  1. Gunduz O, Yurtgezen ZG, Topuz RD, Sapmaz-Metin M, Kaya O, Orhan AE, Ulugol A. The therapeutic effects of transferring remote ischemic preconditioning serum in rats with neuropathic pain symptoms. Heliyon 2023 Oct;9(10):e20954.
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