The overall goal of this procedure is to observe the activation of the immune system during skin infections by injecting Candida albicans to preserve skin architecture and the localization of specific immune and non-immune cells. This method can help answer key question in the immunological field such as the nature and the localization of the immune cells recruited during skin infections and the principal mediators involved. The main advantage of this technique is the maintenance of the structural architecture of the skin after fungal injection that allows us to understand the localization of each immune cellular type.
Demonstrating part of the procedure will be Giulia Stamerra, a PhD student from the laboratory of Professor Marina Vai. To begin, inoculate C.Albicans strain CAF3-1 in tubes containing rich medium supplemented with 50 milligrams per liter of uridine and culture at 25 degrees Celsius. Under these condition, C.albicans grows as a yeast.
Monitor the culture by using a cell counter analyzer or a spectrophotometer to measure the cellular concentration. When the culture reaches eight times 10 to the sixth cells per milliliter, harvest the sample. Use YEPD-uridine medium with Hepes as a buffering agent to re-suspend the cells and incubate them at 37 degrees Celsius to induce hyphae formation.
Following five hours in culture, when hyphae should be visible, under a microscope at 100X magnification check hyphal formation. Culture the cells for a total of 16 hours, at which time 95%of the culture will have formed hyphae. To further enrich the concentration of hyphae, centrifuge aliquots of the culture at 3, 300 times g for five minutes.
Discard the supernatant and use sterile PBS to re-suspend the pellet at a concentration of one times 10 to the eighth hyphae per milliliter. After anesthetizing a shaved mouse according to the text protocol, check the paw reflex to ensure the animal is deeply sedated. To inject C.albicans hyphae in the deep derma, use two fingers to pull the skin of the shaved flank taut, then insert a 30-gauge eight-millimeter needle attached to a 0.3-milliliter insulin syringe at an angle of 10 to 15 degrees, with the bevel of the needle facing up, and inject a final volume of 50 microliters corresponding to five times 10 to the sixth hyphae.
If the angle of the injection is higher than 15 degrees, the pathogen will be injected too deep into the derma, risking damage to the peritoneum and, above all, losing the possibility to study infections specifically at the skin level. At this angle, the pathogen will be injected at a depth of approximately 300 to 500 microns. To ensure that the injection is well performed, check that the injected volume forms a bump that is absorbed after a few minutes.
For time points of less than 24 hours, use a marker pen to mark the area of infection, since the bump formed with the injection persists for a few minutes. For time points of 24 hours or later, this step is not required, because either an ulcer or a cyst will be clearly visible. After euthanizing the mice using institutional guidelines, with surgical forceps pull the skin at the border of the injected site previously marked with the marker pen, then, using surgical scissors, excise the infected site by cutting the skin following the marked line.
Immerse the skin with the internal side facing up in a disposable base mold filled with O.C.T.compound. Freeze the sample in liquid nitrogen until the compound becomes white. Do not let the liquid nitrogen cover the sample before it is completely frozen, as this will damage the sample.
To prepare slides, starting from the center of the sample cut the sample in half to prepare the slices for histology, then, using a cryostat, cut five-micron thick slices. Use positively-charged microscope slides to collect the slices. To carry out H&E staining, immerse the slides in Gill's hematoxylin for four minutes, then wash the slides in running tap water for five minutes.
Immerse the slides in eosin-Y solution for one minute, wash the slides in running tap water for five minutes, then use distilled water to rinse the slides. Next dehydrate the samples by immersing them in an ethanol series for 15 seconds each. To clear the slides, transfer them to a histological clearing agent for at least two minutes, then use mounting medium and cover slips to mount the samples.
Use a microscope slide scanner at 40X magnification to image the slides. Perform Period Acid Solution or PAS staining by using room-temperature acetone to fix the slides for one minute, then use running tap water to wash the slides for one minute. Immerse the slides in PAS for five minutes, then use three changes of distilled water to wash the samples.
Next immerse the slides in Schiff's reagent for 15 minutes, then use running tap water to wash the slides for five minutes. Immerse the sample in Gill's hematoxylin for five minutes to stain them, then wash the slides in running tap water for 30 seconds. Dehydrate the samples by immersion in three changes of 100%ethanol for 15 seconds each, then immerse the slides in a histological clearing agent.
With mounting medium and cover slips, mount the cleared samples, then use a microscope slide scanner to image the samples. After euthanizing the mice and removing skin samples as demonstrated earlier in this video, immerse the samples in a two-milliliter snap-cap tube with one milliliter of reagent for acid guanidinium thiocyanate phenol chloroform extraction. Use surgical forceps to cut the tissue into 0.2-square centimeter pieces.
With a bead mill, smash the samples at 20 oscillations per second for 20 minutes. If intact pieces of skin are still present, repeat the smashing until the sample is completely homogenized. Centrifuge the samples at 16, 000 times g for one minute.
Keep the supernatants for RNA extraction and discard the pellet. This step is performed to eliminate hair and debris that could interfere with the extraction process. After extracting the RNA with a purification column, use a spectrophotometer to measure the RNA concentration and purity.
The maintenance of skin structural integrity allows the detection of immune cells and their localization at the site of infection. This high magnification reveals that the abcess is mainly composed of PMCs that contain the spread of the pathogen by its confinement to the site of infection. The surrounding areas of the abcess are also enriched in PMCs.
In this figure, PAS staining, which gives the fungus a purple-magenta color, clearly shows that the pathogen is confined inside the abcess formed by the recruitment of granulocytes. C.albicans is clearly visible at higher magnification and it appears to be surrounded by necrotic and immune cells. At the beginning of the infection process, immune cell recruitment leads to the formation of the abcess.
48 to 72 hours after infection, the disruption of the skin structures and the formation of a scar were visualized due to the healing phase following expulsion of the pathogen. As shown here, wild type mice display an ulcerative process that increases over time. Upon specific treatments or genetic deficiencies, the formation of the ulcer can be reduced and/or abrogated.
In some cases, instead of an ulcer, a cyst will form as seen here. Following this infection procedure, other methods like protein extractions can be performed in order to answer additional questions like the activation of specific intracellular pathways. After watching this video, you should have a good understanding on how to perform injection of C.Albicans in the skin to study the involvement of the immune system during microbial infection without disrupting the architecture of the epithelium.
Don't forget that working with reagents for RNA extractions can be extremely hazardous and precautions such as using a fume hood should always be taken while performing this procedure.
