Gamma irradiation is a widely used treatment for colorectal cancer. And while it is therapeutically efficacious, it has negative effects on gastrointestinal tract. The presented protocol describes an efficient method to study the activation, differentiation, and migration of cells during regeneration after radiation injury.
This protocol describes a robust and reproducible radiation injury model. Importantly, this model allows to track intestinal cell fate following injury and thus provides better understanding of regenerative capacity of specific cell subpopulation. This technique can employ distinct lineage-tracing animal models to study the realtime behavior and the interaction of various subpopulations of cells post-injury.
Minor modifications to this protocol should allow for the investigation of the relationships between microbiota and different epithelial, stromal, and immune cell populations upon radiation injury. To begin, resuspend tamoxifen powder in sterile corn oil at a concentration of 30 micrograms per microliter. Sonicate for three minutes in 30 seconds on-off cycles with 60%amplitude.
Mix by rotation in the dark for one hour at room temperature. Then, prepare EdU by adding sterile DMSO to EdU powder. Add 1/5 of the total volume, and slowly add the remaining volume of sterile ultrapure water.
When completely dissolved, aliquot and store at 20 degrees Celsius. Prepare reagents for tissue collection and fixation, such as 70%ethanol in water, DPBS chilled to four degrees Celsius, Bouin's fixative buffer, and 10%buffered formalin. Next, prepare the necessary equipment, such as a mice dissection kit, Petri dishes, a 16-gauge gavage needle attached to a 10-milliliter syringe and histological cassettes.
Two days prior to gamma irradiation exposure, prepare the experimental animals for tamoxifen injection. Weigh each animal, and calculate the required dosage. Disinfect the abdominal area with 70%ethanol.
Then, administer a single dose of tamoxifen intraperitoneally using a 27-gauge needle attached to a one-milliliter syringe to induce Cre-mediated recombination. Observe animals for the next 48 hours to exclude potential tamoxifen toxicity. After 48 hours, transfer the animals to the irradiation room.
Disinfect the sample chamber in the gamma irradiator with 70%ethanol solution. Place the absorbent mat and animals inside the sample chamber. Place the lid, and close the chamber.
Program the gamma irradiator to expose animals to 12 gray total body irradiation. Then, turn the key to the start position, and press start to initiate the exposure. Leave the room for active exposure time.
After the preset time has elapsed, the machine will stop automatically and will start beeping. Turn off the machine by turning the key into the stop position. Open the sample chamber, and take off the lid.
Transfer the animals to the cage, and disinfect the sample chamber with 70%ethanol. Transfer the animals back to the conventional housing room, and observe their condition post-treatment. Monitor the weight of the animals every day.
Three hours prior to the planned euthanasia, disinfect the abdominal area, and inject the mice intraperitoneally with 100 microliters of EdU stock solution using a 28-gauge insulin syringe. After euthanizing the animal, collect the proximal intestines at different time points post-irradiation. Dissect the proximal part of the small intestine, and remove attached tissues.
Flush the tissue with cold DPBS using a 16-gauge straight feeding needle attached to a 10-milliliter syringe, and fix the tissue with Bouin's fixative buffer. Cut the intestines open longitudinally, and roll the proximal intestines using the Swiss-roll technique. Place the tissues in a histological cassette in a container with 10%buffered formalin, and incubate it for 24 to 48 hours at room temperature.
After incubation, transfer the histological cassettes to 70%ethanol, and embed the tissue in paraffin. After embedding, place the histological cassettes on ice for at least one hour. Then, cut five-micron-thick horizontal sections using a microtome.
Transfer the tissue sections to a water bath warmed up to 45 degrees Celsius, and place the sections on charged slides. Leave the slides on a rack overnight at room temperature to dry. Hematoxylin and eosin images of small intestine specimens showed a homeostatic intestinal epithelium at zero hour, followed by the loss of cells within crypt compartments during the apoptotic phase.
This was followed by the regenerative phase in which highly proliferative cells populated the crypts, leading to the normalization phase by day seven. Lineage tracing by EYFP showed that during homeostasis, the cells originating from Bmi1 positive reserve stem cells were restricted to the plus four to plus six positions within crypts. EdU and Ki-67 staining showed normal intestinal homeostasis in sham irradiated mice, extending from active stem cells through the transit-amplifying zone.
During the apoptotic phase, the number of EYFP-positive cells decreased, accompanied by a loss of proliferative cells caused by radiation damage. In the regenerative phase, activation of reserve stem cells and rapid proliferation of Bmi1-positive cells was observed. Further proliferation and migration restored intestinal epithelium integrity.
TUNEL staining showed that apoptotic cells are absent during homeostasis. An increase in TUNEL staining was observed late in the apoptotic phase. During the regenerative phase, TUNEL staining steadily decreased within the regenerating crypts and was nearly absent when the crypts normalized.
This technique can be combined with RNA sequencing, single-cell RNA sequencing, fluorescence-activated cell sorting, or proteomic analysis to glean more in-depth knowledge about the role of specific cell lineages during intestinal regeneration following injury. The researchers can investigate the interactions between microbiota and epithelial, stromal, and immune cell populations in gut regeneration to expand the understanding of the regenerative capability of the intestinal epithelium.
