Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses
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 dis...
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| Format: | Article |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2022.894189/full |
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| author | Nina Höglund Ninna Koho Heini Rossi Jenni Karttunen Anne-Mari Mustonen Anne-Mari Mustonen Petteri Nieminen Kirsi Rilla Sanna Oikari Anna Mykkänen |
| author_facet | Nina Höglund Ninna Koho Heini Rossi Jenni Karttunen Anne-Mari Mustonen Anne-Mari Mustonen Petteri Nieminen Kirsi Rilla Sanna Oikari Anna Mykkänen |
| author_sort | Nina Höglund |
| collection | DOAJ |
| description | 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, n = 35) and healthy horses (n = 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 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 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. |
| first_indexed | 2024-04-12T13:41:11Z |
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| institution | Directory Open Access Journal |
| issn | 2297-1769 |
| language | English |
| last_indexed | 2024-04-12T13:41:11Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Veterinary Science |
| spelling | doaj.art-862bd0a774c249bfb0bcd26771ea3d552022-12-22T03:30:51ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692022-06-01910.3389/fvets.2022.894189894189Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic HorsesNina Höglund0Ninna Koho1Heini Rossi2Jenni Karttunen3Anne-Mari Mustonen4Anne-Mari Mustonen5Petteri Nieminen6Kirsi Rilla7Sanna Oikari8Anna Mykkänen9Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, FinlandDepartment of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, FinlandDepartment of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, FinlandDepartment of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, FinlandInstitute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, FinlandDepartment of Environmental and Biological Sciences, Faculty of Science and Forestry, University of Eastern Finland, Joensuu, FinlandInstitute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, FinlandInstitute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, FinlandInstitute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, FinlandDepartment of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, FinlandExtracellular 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, n = 35) and healthy horses (n = 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 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 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.https://www.frontiersin.org/articles/10.3389/fvets.2022.894189/fullairway diseasebronchoalveolar lavage fluid (BALF)equine asthmaextracellular vesicle (EV)inflammationsize-exclusion chromatography |
| spellingShingle | Nina Höglund Ninna Koho Heini Rossi Jenni Karttunen Anne-Mari Mustonen Anne-Mari Mustonen Petteri Nieminen Kirsi Rilla Sanna Oikari Anna Mykkänen Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses Frontiers in Veterinary Science airway disease bronchoalveolar lavage fluid (BALF) equine asthma extracellular vesicle (EV) inflammation size-exclusion chromatography |
| title | Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses |
| title_full | Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses |
| title_fullStr | Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses |
| title_full_unstemmed | Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses |
| title_short | Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses |
| title_sort | isolation of extracellular vesicles from the bronchoalveolar lavage fluid of healthy and asthmatic horses |
| topic | airway disease bronchoalveolar lavage fluid (BALF) equine asthma extracellular vesicle (EV) inflammation size-exclusion chromatography |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2022.894189/full |
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