Immunology and Infection
Published: September 18th, 2021
We describe the method for quantitative analysis of the distribution of Aspergillus fumigatus conidia (3 µm in size) in the airways of mice. The method also can be used for the analysis of microparticles and nanoparticle agglomerate distribution in the airways in various pathological condition models.
Aspergillus fumigatus conidia are airborne pathogens that can penetrate human airways. Immunocompetent people without allergies exhibit resistance and immunological tolerance, while in immunocompromised patients, conidia can colonize airways and cause severe invasive respiratory disorders. Various cells in different airway compartments are involved in the immune response that prevents fungal invasion; however, the spatio-temporal aspects of pathogen elimination are still not completely understood. Three-dimensional (3D) imaging of optically cleared whole-mount organs, particularly the lungs of experimental mice, permits detection of fluorescently labeled pathogens in the airways at different time points after infection. In the present study, we describe an experimental setup to perform a quantitative analysis of A. fumigatus conidia distribution in the airways. Using fluorescent confocal laser scanning microscopy (CLSM), we traced the location of fluorescently labeled conidia in the bronchial branches and the alveolar compartment 6 hours after oropharyngeal application to mice. The approach described here was previously used for detection of the precise pathogen location and identification of the pathogen-interacting cells at different phases of the immune response. The experimental setup can be used to estimate the kinetics of the pathogen elimination in different pathological conditions.
On a daily basis, people inhale airborne pathogens, including spores of opportunistic fungi Aspergillus fumigatus (A. fumigatus conidia) that can penetrate the respiratory tract1. The respiratory tract of mammals is a system of airways of different generations that are characterized by the different structures of the airway walls2,3,4. Tracheobronchial walls consist of several cell types among which are ciliated cells that provide the mucociliary clearance5. In the alveoli, there are no ciliated cells and the p....
All methods concerning laboratory animals described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (protocol number 226/2017).
1. A. fumigatus conidia application
Following the protocol above, the 3D image showing the airways and A. fumigatus conidia in the lung lobe of a mouse was obtained (Figure 1A). Streptavidin (that was used for airway visualization) labeled bronchi and bronchioles15. Additionally, the large vessels, which are easily distinguishable from the airways by their morphology, and pleura are visualized in the airway channel (Figure 1A-C). The creation of th.......
Whole-organ 3D imaging permits obtaining of the data without dissection of the specimen, which is of great importance for investigating the spatial aspects of the anatomical distribution of the pathogen in the organism. There are several techniques and modifications of tissue optical clearing that help to overcome the laser light scattering and allow whole-organ imaging15,16,18,19. One of the c.......
The authors thank Prof. Sven Krappmann (University Hospital Erlangen and FUA Erlangen-Nürnberg, Germany) for providing the Aspergillus fumigatus conidia strain AfS150. The authors thank MIPT Press Office. V.B. acknowledges the Ministry of Science and Higher Education of the Russian Federation (#075-00337-20-03, project FSMG-2020-0003). The work regarding A. fumigatus conidia imaging and quantification was supported by RSF № 19-75-00082. The work regarding airways imaging was supported by RFBR № 20-04-60311.....
|Alexa Fluor 594 NHS Ester
|Aspergillus fumigatus conidia
|The strain AfS150, a ATCC 46645 derivative
|Pushchino Animal Breeding Centre (Russia)
|Male. 12 - 30 week old.
|Cell imaging coverglass-bottom chamber
|4 or 8 well chamber with coverglass bottom
|Any centrifuge provided 1000 g can be used
|Confocal laser scanning microscope
|FIJI image processing package
|B. Braun Aesculap
|B. Braun Aesculap
|With groung-in lid
|Graphic Editor Photoshop
|Adobe Photoshop CS
|Imaris Microscopy Imaging Software
|Free trial is avalable https://imaris.oxinst.com/microscopy-imaging-software-free-trial
|Plan-Apochromat, 10 × (NA = 0.3)
|5 to 50 μL
|Streptavidin, Alexa Fluor 488 conjugate
|Tris (hydroxymethyl aminomethane)
|Mr 121.14; CAS Number: 77-86-1
|ZEN microscope software
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