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Frontiers in veterinary science2022; 9; 894189; doi: 10.3389/fvets.2022.894189

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses.

Abstract: Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell-cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, = 35) and healthy horses ( = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 10 ± 1.42 × 10 particles/mL in the EA horses and 1.33 × 10 ± 1.07 × 10 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.
Copyright © 2022 Höglund, Koho, Rossi, Karttunen, Mustonen, Nieminen, Rilla, Oikari and Mykkänen.
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Publication Date: 2022-06-21 PubMed ID: 35799843PubMed Central: PMC9255554DOI: 10.3389/fvets.2022.894189Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research studies the extraction of extracellular vesicles, or EVs, from bronchoalveolar lavage fluid, obtained from healthy and asthmatic horses. The researchers compare the number and size of these particles to further understand their role in inflammatory reactions, with potential implications for studying airway inflammation in horses.

Understanding the Objectives

  • The primary goal of this research was to reveal the quantity and size distribution of extracellular vesicles (EVs) found in the bronchoalveolar lavage fluid (BALF) of healthy and asthmatic horses.
  • Extracellular vesicles are minuscule membrane-bound particles involved in inflammatory responses, usually by assisting cellular interactions.
  • Information obtained from this research may lead to an improved comprehension of EVs role in airway inflammation, particularly in horses.

Exploring the Methodology

  • The researchers studied the bronchoalveolar lavage fluid (BALF) of 35 asthmatic horses and 19 healthy horses. This fluid was collected during bronchoscopy, where saline was injected into the right lung and manually aspirated soon after.
  • After retrieval, the fluid underwent two instances of centrifugation to eliminate cells and biological debris, leaving primarily extracellular vesicles. This supernatant fluid was then concentrated, and the EVs isolated via size-exclusion chromatography.
  • They used nanoparticle tracking analysis (NTA) to determine the quantity and scale of these vesicles.
  • The researchers also utilized transmission electron microscopy and confocal laser scanning microscopy to visually confirm and track the extracellular vesicles.

Key Results and Observations

  • The researchers successfully isolated and preserved EVs with the mean size of about 247 ± 35 nm in asthmatic horses and 261 ± 47 nm in the healthy control horses.
  • They found the average concentration of EVs to be 1.38 × 10 ± 1.42 × 10 particles/mL in asthmatic horses and 1.33 × 10 ± 1.07 × 10 particles/mL in the controls, with no significant statistical difference between the two groups.
  • Through Western blotting and microscopy methods, these particles were shown to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan.
  • The researchers concluded that the equine BALF is rich in EVs of various sizes, and the described collection protocol can be efficiently applied to isolate them.

Future Perspectives and Implications

  • While this study does not show significant differences in EVs between healthy and asthmatic horses, the methodology developed here provides a means to study EVs in more depth and could potentially be used to understand and study their involvement in airway inflammation in horses.

Cite This Article

APA
Höglund N, Koho N, Rossi H, Karttunen J, Mustonen AM, Nieminen P, Rilla K, Oikari S, Mykkänen A. (2022). Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses. Front Vet Sci, 9, 894189. https://doi.org/10.3389/fvets.2022.894189

Publication

Frontiers in veterinary science
ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 9
Pages: 894189
PII: 894189

Researcher Affiliations

Höglund, Nina
  • Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
Koho, Ninna
  • Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
Rossi, Heini
  • Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
Karttunen, Jenni
  • Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
Mustonen, Anne-Mari
  • Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
  • Department of Environmental and Biological Sciences, Faculty of Science and Forestry, University of Eastern Finland, Joensuu, Finland.
Nieminen, Petteri
  • Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
Rilla, Kirsi
  • Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
Oikari, Sanna
  • Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland.
Mykkänen, Anna
  • Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Citations

This article has been cited 2 times.
  1. Höglund N, Nieminen P, Mustonen AM, Käkelä R, Tollis S, Koho N, Holopainen M, Ruhanen H, Mykkänen A. Fatty acid fingerprints in bronchoalveolar lavage fluid and its extracellular vesicles reflect equine asthma severity. Sci Rep 2023 Jun 17;13(1):9821.
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  2. Hickman E, Carberry V, Carberry C, Cooper B, Mordant AL, Mills A, Sokolsky M, Herring LE, Alexis NE, Rebuli ME, Jaspers I, Sheats K, Rager JE. Respiratory extracellular vesicle isolation optimization through proteomic profiling of equine samples and identification of candidates for cell-of-origin studies. PLoS One 2025;20(1):e0315743.
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