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

This research aims to establish an efficient protocol for preserving the airway contents of mice in lung infection models. We're developing the capacity to answer the question of where inhaled fungal spores land in the lungs. Recently, many groups have identified roles for various airway epithelial cells in sensing infections and initiating protective immune responses.

These epithelial cells range from club cells in bronchials to type II epithelial cells in the terminal alveoli. The common lung preparation technique is to inflate the lung with the liquid fixative to preserve the lung tissue for downstream histological analyses. However, this technique displaces contents of the airways, such as inhaled fungal spores.

Our protocol preserves the natural location of inhaled fungal spores in the lung while maintaining proper lung morphology via air inflation and proper lung fixation through vascular perfusion. We hope to use this protocol to characterize the bronchoalveolar junctions where spores appear to be landing in our model, but we'll utilize the locations of the spores in proximity to various epithelial cells and study the resulting immune responses. To begin, stain one times 10 to the power of six Coccidioides posadasii spores with five micromolar concentration of CFSE for 30 minutes at 30 degrees Celsius.

After staining, wash the spores with PBS. Centrifuge the spores at 12, 000 g and resuspend the pellet in 25 microliters of PBS for spore inoculum at the appropriate concentration. Prepare a pipette with 25 microliters of the spore inoculum.

After anesthetizing the mouse in a Nalgene jar, position the mouse in a supine position in one hand and allow it to take three to five regular gasping breaths. Place the pipette tip at the posterior of the mouse oropharynx and release the inoculum during inhalation. Feel crackles on the posterior and anterior aspects of the mouse thorax with hand and thumb to confirm inhalation of the inoculum.

Spray the deeply anesthetized mouse with 70%ethanol. Using scissors, carefully snip the skin of the mouse, then pull it apart to expose the peritoneum. Then, pull the skin from the superior half over the mouse's head, freeing the arms.

Now, cut the peritoneal membrane at the sternum and along the inferior aspect of the ribcage, exposing the peritoneum. Displace the liver inferiorly to reveal the diaphragm. Use scissors to carefully puncture the diaphragm away from the lung parenchyma.

Next, cut the ribcage on the left side to expose the heart. Using a 30-gauge needle, inject five to 10 milliliters of PBS into the right ventricle. After removing the needle, use a new needle to inject five milliliters of 10%neutral buffered formalin into the right ventricle.

Using scissors, remove the anterior half of the ribcage. To expose the trachea, cut the superior ribs and collarbones to the right and left of the neck, avoiding the midline where the trachea lies. Using forceps, clasp the remaining top ribs and collarbones near the neck midline and pull superiorly until the trachea is exposed.

Prepare a 10-centimeter suture thread and pull it under the trachea. Pre-tie a loose knot at the inferior end of the exposed trachea. Then, prepare a one-milliliter syringe of air with an 18-gauge catheter.

Using an 18-gauge needle, make a hole in the trachea at the superior end of the exposed region. Now, insert the catheter into the hole, ensuring a tight fit to prevent air from escaping. Then, slowly inject one milliliter of air into the lungs over 10 seconds, watching for the inflation of all lung lobes.

Full inflation results in the lungs wrapping slightly around the heart and filling the volume occupied in the unpunctured diaphragm. After isolating the lungs from the mouse, place it on the top edge of a 50-milliliter conical tube filled with 20 milliliters of 10%neutral buffered formalin. Place the suture threads outside of the tube, then screw down the cap.

To begin, rinse the isolated mouse lungs in PBS, and place them in a 30%sucrose PBS solution for 72 to 96 hours at four degrees Celsius. Afterward, remove excess tissue and place the lungs in a Cryomold with OCT medium. Freeze the specimen at minus 80 degrees Celsius.

Equilibrate the specimen in OCT cryoblocks at minus 20 degrees Celsius for one hour before sectioning the blocks at 20 to 100 micrometers thickness. Collect sections onto a glass slide and allow them to dry for 30 minutes to one hour. Place the slide into a PBS bath at room temperature for 30 minutes to remove OCT from the tissue.

Then, use a wipe to remove any excess droplets from the slide's perimeter around the tissue specimen. Draw a perimeter around the specimen on the slide using a hydrophobic PAP pen and allow it to dry for five minutes. Next, add 300 microliters of freshly prepared animal-free blocking solution onto the slide and incubate at room temperature.

Now, incubate the slide in 300 microliters of primary Alexa Fluor 647 conjugated rat anti-mouse EpCAM antibody solution overnight at four degrees Celsius. The next day, remove the antibody solution and wash the slide twice with 300 microliters of PBS for five minutes. After drying, add one drop of room temperature soft-set SlowFade glass mounting medium to each piece of tissue.

Place a coverslip over the tissue, ensuring it extends beyond the sample and the hydrophobic marker perimeter. To begin, place the fungal spore inoculated, immunostained mouse lung section under a multi-channel fluorescent microscope and select an appropriate objective. To capture the entire lung lobe, collect sufficient image tiles and merge them together.

Using the microscope's software, export the image of the entire lung lobe for downstream processing. In QuPath, create a project file and add the fluorescent images to the file. Classify the EpCAM-positive pixels as epithelium by sequentially selecting Classify, Pixel classification, and Create thresholder.

Select the resolution, channel, smoothing sigma, and threshold value to identify the target region. Save the classifier under a unique name and select Create objects. In the new Create objects window, select New object type as Annotation.

For lung epithelium, set the minimum object size and minimum hole size to 100 square micrometers. After selecting OK, the annotations will be created and can be modified using the brush tool. To classify spores, repeat the steps as demonstrated for lung epithelium annotations.

Then, from the Create objects window, select Detection as the new object type. Set the minimum object size and minimum hole size to zero square micrometers and select Split objects. Next, to conduct spatial analysis of the spores sequentially, select Analyze, Spatial analysis, and calculate signed distance to annotations two-dimensional.

The distances to EpCAM-positive epithelium will be calculated for each detected spore. Finally, click Measure, followed by Export measurements to export the results. Spores of Coccidioides posadasii accumulated primarily in distal bronchials and nearby alveolar spaces in the air-inflated lungs.

Spores appeared more dispersed away from bronchials in formal and liquid-inflated lungs compared to air-inflated lungs. The distance between spores and EpCAM-positive bronchiolar epithelium was greater in liquid-inflated lungs compared with physiologic air inflation, indicating more dispersed spores.