Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The overall goal of this protocol is to quantify the pulmonary fungal burden in mice with invasive aspergillosis by histological analysis. This method can help answer key questions in the fungal pathogenesis field about protective and detrimental host response mechanisms and key fungal virulence factors. The main advantage of this technique is that existing histological samples can be used to obtain fungal burden with comparable results to quantitative PCR application of lung fungal DNA.

Although this method can provide insight into invasive aspergillosis, it can also be applied to other disease studies in which pathogens can be differentially stained within host tissues. We first had the idea for this method when we began thinking of alternative methods for quantifying lung fungal burden with less variability than DNA quantitative methods. Demonstrating the infection and harvest procedures will be Angar Tsoggeral, a research technician.

24 hours before the infection, deplete the neutrophils in each experimental seven-to-10-week-old mouse with 0.5 milligrams of anti-mouse-Ly-6G antibody in sterile saline delivered intraperitoneally at a 45-degree angle into the lateral lower right quadrant of each animal and return the mice to their cages. The next morning, pour 1.5 grams of 0.5-millimeter glass beads onto an A.fumigatus conidia culture plate and gently tilt the plate back and forth until the beads are coated with the fungus. Collect the conidia-coated bead mixture into a 15-milliliter conical tube and suspend the beads in five milliliters of DPBS.

Vortex the beads to detach them from the fungus and count the conidia in a 50X dilution of supernatant on a hemocytometer. Dilute the conidia to a one times 10 to the 8th conidia per milliliter of DPBS concentration and load 50 microliters of conidia solution per mouse into individual micropipettes. After confirming a lack of response to toe pinch, place the first anesthetized mouse on a slant board in the supine position and gently restrain the upper incisors with a rubber band and the lower incisors with a metal wire.

Then, use small forceps to carefully hold the tongue at full extension and deliver 50 microliters of the conidia solution to the base of the tongue. Keeping the tongue at full extension, listen for rapid breathing and the distinct clicking noise of aspiration for 20 seconds or until the suspension is fully aspirated. Then, gently return the mouse to its cage with monitoring until full recumbency and infect the next animal.

When all of the mice have been infected, inject a subset of the animals with an antifungal agent and repeat the neutrophil depletion in all of the animals 24 hours post-infection. Three days after administering the fungus, place the first mouse in the supine position on a foam board covered with a absorbent pad. Ensure the mouse is euthanized with a toe pinch, and disinfect the hair with 70%ethanol.

After securing the limbs, use scissors to carefully open the upper chest cavity cutting away the rib cage to expose the lungs and heart. Cut the inferior vena cava descending from the heart taking care to avoid cutting other blood vessels or puncturing any organs, and use a five-milliliter syringe equipped with a 25-gauge needle held at a 45-degree angle to slowly perfuse five milliliters of cold DPBS into the right ventricle of the heart. After perfusion with five milliliters of 10%formalin in the same manner, carefully expose the trachea and detach the surrounding fat pads taking care not to puncture the trachea.

Elevate the exposed trachea with forceps and use a 25-gauge needle to poke a hole into the upper trachea. Carefully insert a cannula through the hole towards the lungs and tie a surgical thread loosely around the trachea to secure the cannula. Use a one-milliliter syringe to inflate the lungs with one milliliter of 10%formalin buffer through the cannula and quickly, but carefully, remove the cannula.

Tighten the surgical thread to seal off the trachea and gently pull on the thread to disconnect the connective tissue allowing the lungs and trachea to be harvested. Place the tissues in a 15-milliliter conical tube containing 15 milliliters of 10%formalin buffer with one end of the thread outside of the tube. Then, seal the cap and invert the tube to completely submerge the sample and fixative for at least 24 hours.

To image the harvested tissue, first open an appropriate imaging quantification program and use a light microscope to obtain at least four distinct fields of each Gomori's modified methanamine silver, or GMS-stained slide, under the 10X objective. Ensure that the images are representative of the infected airways within the lung and that they exhibit a similar tissue density. To add scale bars to the images, under the Edit menu, select Add/Edit Calibration Marks and select the line thickness, color and scale bar size.

Click on the image where the scale bar is to be placed, then open the Edit menu and select Merge and Merge Calibration Marks to add a scale bar to each image and save the images into the appropriate experimental group folders. To calculate the fungal burden in each animal, open an appropriate image processing program and open the first folder of images. Under the Image menu, select Type and RGB Stack to convert the images.

Using the right arrow key, select the second of the three images and depress Control-Shift-T to open the Threshold menu settings adjuster. Locate the original tiff or jpg image as a reference and open the image with an image viewing program. When selecting the threshold value, it is critical to be able to view the original image so that the selected area is representative of the fungal hyphae and to ensure consistency between samples.

Move the top slider all the way to the left and adjust the bottom slider until the selected red area is representative of the microscopy image. Click Set and OK, and select Analyze and Set Measurements checking Area, Area fraction, Limit to threshold, Display label and leaving the bottom settings as default. Click OK and select Analyze and Measure.

The results should appear. Then, export the data to a data analysis program for graphing and statistical analysis and enter the mean of the four or more area percentages for each lung sample. As this representative survival graph demonstrates, eosinophil-deficient mice with invasive aspergillosis treated with an antifungal agent post-infection exhibit an increased survival compared to wild-type animals.

Representative GMS staining from neutropenic wild-type and eosinophil-deficient mice with invasive aspergillosis reveals a near-total fungal clearance in antifungal-treated knockout mice that is not reciprocated in wild-type animals with or without antifungal treatment. Further, quantitative PCR and manual light microscopy GMS quantification as just demonstrated both confirm that antifungal agent treatment results in the most significant fungal burden decrease between wild-type and eosinophil-deficient mice. However, in untreated mice, only GMS quantification determined a significant decrease in eosinophil-deficient mice in this experiment.

When the mean areas of whole lung GMS stained sections are calculated under four-X magnification, the differences are similar to the results obtained with four representative 10X fields although with less statistical significance. Similar survival, fungal burden and fungal clearance results are also observed in gamma-delta T-cell-deficient mice treated with the same antifungal agent. While attempting this procedure, it is important to remain consistent when taking pictures of the histological sections and when adjusting the threshold value to obtain comparable results.

Following this procedure, other methods involving the histological analysis of samples of clear differences between target and background staining can be performed to answer additional questions related to disease pathology in the lungs and other tissues. After watching this video, you should have a good understanding of how to infect mice with fungal conidia by involuntary aspiration and how to measure fungal growth in lung tissue sections with an image processing software. Don't forget that working with Aspergillus fumigatus and Gomori's modified methanamine silver stain can be hazardous and that precautions such as wearing personal protective equipment and working under a well-ventilated hood should always be taken while performing this procedure.