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, 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.

Fatty acid fingerprints in bronchoalveolar lavage fluid and its extracellular vesicles reflect equine asthma severity

“…Arterial blood sampling, airway endoscopy, collection of BALF, and cytology were performed as described previously using detomidine (0.01 mg/kg, Domosedan, Orion, Espoo, Finland) and butorphanol (0.005–0.01 mg/kg, Butordol, Intervet, Boxmeer, the Netherlands) for sedation and 1% lidocaine solution (20–40 mL, Lidocain, Orion) for local anesthesia 27 . Collection of BALF was performed with sterile 0.9% saline by injecting 360 mL to horses and 240 mL to ponies in one aliquot, followed by manual aspiration of the fluid.…”

“…For SUP, BALF was centrifuged at +4°C, 300 × g for 10 min followed by centrifugation of the supernatant at 3000× g for 20 min. The EV population (particle size up to 1000 nm) was isolated and purified with size-exclusion chromatography (SEC), as described previously 27 . One control horse did not have a SUP sample, and therefore the sample sizes were 30 BALF samples, 29 SUP samples, and 30 EV samples.…”