Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

Summary

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Abstract

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3Δ arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces.

Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

Introduction

Humans can inhale up to billions of spores per day from a variety of environmental fungi¹. To understand our barrier defenses against these inhaled spores, we must appreciate the precise microanatomical environments where these spores land within the airways and lung parenchyma. The cellular composition of the airways (i.e., epithelial cells) significantly transforms along the trachea, bronchi, bronchioles, and alveoli. Each of these distinct regions is composed of different cell types with discrete functions which avails an arsenal of defenses to prevent pathologic infection.

The precise location of pulmonary fungal....

Protocol

All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) of Rutgers Biomedical Health Sciences.

1. Label spores with intracellular fluorescence

1. Stain 1 x 10⁶ spores in carboxyfluorescein succinimidyl ester (CFSE), Cell Tracker Orange, or Cell Tracker Red (or intracellular dye of choice) at 5 µM for 30 min at 30 °C, then wash with PBS, and spin the spores at 12,000 x g. Repeat .......

Discussion

We have established a pipeline for spore inhalation and analysis of the spatial deposition of the inhaled spores. This pipeline provides valuable information to determine the relevant stromal regions of the lung affected by inhalation of Coccidioides posadasii cts2/ard1/cts3Δ arthroconidia. We have observed that Coccidioides spores and similarly sized inert particles (data not shown) accumulate in distal bronchiolar regions and broncho-alveolar junctions rather than fully dispersed throughout alve.......

Materials

Name	Company	Catalog Number	Comments
18 G, 1 1/2 needle (305185)	Fisher	305185
1 L  Screwtop Jar  (Nalgene)	Fisher Scientific	11-823-33
Air-Tite Bulk Unsterile Syringes 10 mL Luer Lock	Fisher	14-817-175
AnaSed Injection (xylazine sterile solution)	Akorn	59399-110-20
Animal-Free Blocker and Diluent, R.T.U.	Vector	SP-5035-100
BD Insyte Autoguard Winged Shielded IV Catheter with BD Vialon Catheter Material 18 G x 1.88 in	BD	381547
BD Pharmingen Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™)	BD	553142
CellTracker Orange CMRA Dye	Fisher Scientific	NC0873640
CFSE	Labviva	75003
Coccidioides posadasii cts2/ard1/cts3Δ	BEI Resources	NR-166
Corning 70 micron strainers	VWR	10054-456
EpCAM AlexaFluor647 monoclonal antibody	Biolegend	118211
Exel International HYpodermic Needles 30 G x 1/2"	Labviva	EN3012
Fisherbrand Sterile Syringes for Single Use (1mL, Leur Slip)	Fisher	14-955-462
Glucose Monohydrate	Azer Scientific	ES17530-500G
High Vacuum Grease	VWR	59344-055
Hoechst 33342 Solution 20 mM (5 mL)	ThermoFischer	62249
Isoflurane USP	Covetrus	29405
Ketamine Hydrochloride	Dechra	1000001250
KIMWIPES Delicate Task Wipers (4.4'' x 8.4")	VWR	21905-026
Lexer Baby Scissors	FST	14078-10
Micro-Adson Forceps	FST	11018-12
Neutral Buffered Formalin (10%) (Azer Scientific)	Fisher	22-026-350
Nunc EasYFlask tissue culture flasks, T75, filter caps	VWR	15708-134
PBS	VWR	45000-446
Peel-A-Way embedding molds	Sigma	E6032-1CS
QuPath 0.5.1 Software	Open-source	https://qupath.github.io/
Silk Suture thread size 3/0	FST	18020-30
SlidesMicro Slides Superfrost Plus	VWR	48311-703
SlowFade Glass Soft-set Antifade Mountant (2 mL)	ThermoFischer	S36917
Sucrose	Sigma	S0389-500G
Tissue-Tek O.C.T. Compound, Sakura Finetek	VWR	25608-930
Tween 20	ThermoFischer	J20605.AP
Vector Laboratories ImmEDGE Hydrophobic Barrier Pen Set Of 2	Fisher Scientific	NC9545623
VWR Micro Cover Glasses, Rectangular (24 mm x 40 mm #1.5)	VWR	48393-230
White Plastic Wire Mesh	MAPORCH	789862904922
Yeast Extract	Fisher	BP9727-500
Zeiss AxioScan 7	Carl Zeiss Microscopy GmbH	https://www.zeiss.com/microscopy/us/products/imaging-systems/axioscan-for-biology.html	multichannel fluorescent microscope
ZEN 3.7 Software	Carl Zeiss Microscopy GmbH	https://www.zeiss.com/microscopy/us/products/software/zeiss-zen.html	microscopy software