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JoVE Journal Biology

Biology

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

Published: July 27, 2022 doi: 10.3791/64028
Automatic Translation
1, 1, 1, 1,2, 1
1Department of Medicine, Renaissance School of Medicine at Stony Brook University, 2Department of Physiology and Biophysics, Renaissance School of Medicine at Stony Brook University

Summary

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

Abstract

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

Introduction

The human intestinal epithelium would cover approximately the surface of half a badminton court if placed completely flat1. Instead, this single cell layer separating humans from the contents of their guts is compacted into a series of finger-like projections, villi, and indentations, crypts that maximize the surface area of the intestines. The cells of the epithelium differentiate along a crypt-villus axis. The villus primarily consists of nutrient-absorbing enterocytes, mucus-secreting goblet cells, and the hormone-producing enteroendocrine cells, while the crypts primarily consist of defensin-producing Paneth cells, active and reserve stem cells, and progenitor cells2,3,4,5. Furthermore, the bi-directional communication these cells have with the stromal and immune cells of the underlying mesenchymal compartment and the microbiota of the lumen generate a complex network of interactions that maintains gut homeostasis and is critical to recovery after injury6,7,8.

The intestinal epithelium is the most rapidly self-renewing tissue in the human body, with a turnover rate of 2-6 days9,10,11. During homeostasis, active stem cells at the base of intestinal crypts (crypt base columnar cells), marked by the expression of leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), rapidly divide and provide progenitor cells that differentiate into all other intestinal epithelial lineages. However, owing to their high mitotic rate, active stem cells and their immediate progenitors are particularly sensitive to gamma-radiation injury and undergo apoptosis following irradiation5,12,13,14. Upon their loss, reserve stem cells and non-stem cells (subpopulation of progenitors and some terminally differentiated cells) within intestinal crypts undergo activation and replenish the basal crypt compartment, which can then reconstitute cell populations of the villi and, thus, regenerate the intestinal epithelium15. Using lineage tracing techniques, multiple research groups have demonstrated that reserve (quiescent) stem cells are capable of supporting regeneration upon the loss of active stem cells13,16,17,18,19,20,21,22. These cells are characterized by the presence of polycomb complex protein 1 oncogene (Bmi1), mouse telomerase reverse transcriptase gene (mTert), Hop homeobox (Hopx), and leucine-rich repeat protein 1 gene (Lrig1). In addition, it has been shown that non-stem cells are capable of replenishing intestinal crypts upon injury23,24,25,26,27,28,29,30,31. In particular, it has been shown that progenitors of secretory cells and enterocytes undergo dedifferentiation upon injury, revert to stem-like cells, and support the regeneration of the intestinal epithelium. Recent studies have identified cells expressing multiple markers that possess the capacity of acquiring stem-like characteristics upon injury (such as DLL+, ATOH1+, PROX1+, MIST1+, DCLK1+)32,33,34,35,36. Surprisingly, Yu et al. showed that even mature Paneth cells (LYZ+) can contribute to intestinal regeneration37. Furthermore, in addition to causing apoptosis of intestinal epithelial cells and disrupting epithelial barrier function, irradiation results in dysbiosis of the gut flora, immune cell activation and the initiation of a pro-inflammatory response, and the activation of mesenchymal and stromal cells38,39.

Gamma radiation is a valuable therapeutic tool in cancer treatment, especially so for colorectal tumors40. However, irradiation significantly affects intestinal homeostasis by inducing damage to the cells, which leads to apoptosis. Radiation exposure causes multiple perturbations that slow down a patient's recovery and is marked by mucosal injury and inflammation in the acute phase and diarrhea, incontinence, bleeding, and abdominal pain long term. This panoply of manifestations is referred to as gastrointestinal radiation toxicity. Additionally, radiation-induced progression of transmural fibrosis and/or vascular sclerosis may only manifest years after the treatment38,41. Simultaneous to the injury itself, radiation induces a repair response in intestinal cells that activates signaling pathways responsible for initiating and orchestrating regeneration42. Radiation-induced small bowel disease can originate from pelvic or abdominal radiotherapy provided to other organs (such as cervix, prostate, pancreas, rectum)41,43,44,45,46. Intestinal irradiation injury is, thus, a significant clinical issue, and a better understanding of the resulting pathophysiology is likely to advance the development of interventions to alleviate the gastrointestinal complications associated with radiotherapy. There are other techniques that allow for investigating the regenerative purpose of the intestinal epithelium apart from radiation. Transgenic and chemical murine models to study inflammation and the regeneration thereafter have been developed47. Dextran sodium sulfate (DSS) induces inflammation in the intestine and leads to the development of characteristics similar to those of inflammatory bowel disease48. A combination of DSS treatment with the pro-carcinogenic compound azoxymethane (AOM) can result in the development of colitis-associated cancer48,49. Ischemia reperfusion-induced injury is another method employed to study the regenerative potential of the intestinal epithelium. This technique requires experience and surgical knowledge50. Furthermore, the aforementioned techniques cause different types of injury than radiation and may lead to the involvement of different mechanisms of regeneration. In addition, these models are time-consuming, while the radiation technique is fairly brief. Recently, in vitro methods utilizing enteroids and colonoids generated from the intestine and colon have been used in combination with radiation injury to study the mechanisms of intestinal regeneration51,52. However, these techniques do not fully recapitulate the organ they model53,54.

The protocol presented includes the description of a murine model of gamma-radiation injury in combination with a genetic model that, following tamoxifen treatment, permits tracing of lineages originating from the reserve stem cell population (Bmi1-CreER;Rosa26eYFP). This model utilizes a 12 Gy total-body irradiation, which induces significant enough intestinal injury to activate reserve stem cells while still allowing for the subsequent investigation of intestinal regenerative capability within 7 days of injury55.

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Protocol

All mice were housed in the Division of Laboratory Animal Resources (DLAR) at Stony Brook University. The Stony Brook University Institutional Animal Care and Use Committee (IACUC) approved all studies and procedures involving animal subjects. Experiments involving animal subjects were conducted strictly in accordance with the approved animal handling protocol (IACUC #245094).

NOTE: Mouse strains B6;129-Bmi1tm1(cre/ERT)Mrc/J (Bmi1-CreER) and B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (Rosa26eYFP) were commercially obtained (see Table of Materials) and crossed to obtain Bmi1-CreER;Rosa26eYFP (Bmi1ctrl) mice, as described previously56,57,58.

1. Housing of Bmi1-Cre ER;Rosa26 eYFP mice

  1. Keep the mice in an animal facility at a temperature ranging from 68-72 °F and humidity ranging from 30-70%, in a 12 h/12 h light/dark cycle, with water and normal chow ad libitum.
  2. Prior to the experiments, confirm mice genotypes using a standard PCR genotyping technique57,58.

2. Preparation of animals and materials

  1. Transfer mice to the experimental housing room at least 7 days before any experimentation to let the mice acclimatize.
  2. Match experimental and control animals according to gender and age.
  3. Subject the control animals to tamoxifen-induced Cre-mediated recombination but not to irradiation (sham treatment, 0 Gy) while ensuring that the experimental animals receive the tamoxifen injection and are exposed to gamma irradiation.
  4. Prepare the tamoxifen solution. Resuspend tamoxifen powder in sterile corn oil at a concentration of 30 mg/mL. Sonicate for 3 min in cycles of 30 s ON and 30 s OFF with 60% amplitude and then rotate in the dark for 1 h at room temperature (RT). Tamoxifen solution can be frozen at −20 °C but do not freeze/thaw it more than once. Preferably, prepare a fresh solution each time and do not store it.
    NOTE: Tamoxifen is light sensitive. Wrap it with tin foil during room temperature rotation.
    CAUTION: Tamoxifen is a potentially hazardous substance: Health Hazard (GHS08) and Environment Hazard (GHS09).
  5. Prepare 5-ethynyl-2′-deoxyuridine (EdU) stock solution. Resuspend EdU powder in 1/5 of the total volume of sterile dimethyl sulfoxide (DMSO) and slowly add the remaining 4/5 of the total volume of sterile ultrapure water. When completely dissolved, aliquot and store at −20 °C.
    CAUTION: 5-ethynyl-2′-deoxyuridine (EdU) and dimethyl sulfoxide (DMSO) are potentially hazardous substances: Health Hazard (H340 and H631, and H227, H315, and H319, respectively). EdU may cause genetic defects and is suspected of damaging fertility or unborn children, and DMSO can cause skin and eye irritation.
  6. Prepare reagents for tissue collection and fixation: dilute ethanol to prepare a 70% water solution, cool down DPBS to 4 °C, and prepare modified Bouin's fixative buffer (50% ethanol and 5% acetic acid in distilled H2O) and 10% buffered formalin.
    CAUTION: Ethyl alcohol is a potentially hazardous substance: Health Hazard (H225-H319), flammable (GHS02), and causes acute toxicity (GHS07). Acetic acid is a potentially hazardous substance: Health Hazard (H226-H314), flammable (GHS02), and corrosive (GHS05). Formalin is a potentially hazardous substance: Health Hazard (H350, H315, H317, H318, H370), and corrosive. Formalin may cause cancer, skin and eye irritation, damage to organs, and allergic skin reactions.
  7. Prepare the equipment necessary for euthanasia according to an approved method (CO2 chamber, for instance), a mice dissection kit (scissors, forceps), Petri dishes, a 16 G gavage needle attached to a 10 mL syringe to flush the intestines, and histological cassettes.

3. Total-body gamma irradiation (TBI) and tissue collection

  1. Two days prior to gamma-irradiation exposure, inject the experimental animals with a single dose of tamoxifen to induce Cre-mediated recombination and BMI1/EYFP+ cell linage tracing. Weigh each animal and calculate a dose of 40 mg/kg of body weight of tamoxifen resuspended in corn oil. Disinfect the abdominal area with 70% ethanol and administer tamoxifen intraperitoneally using a 27G needle attached to a 1 mL syringe.
  2. Observe animals for the following 48 h to exclude potential tamoxifen toxicity.
  3. Transfer the mice to the irradiation room as specified by the local institution.
  4. Calculate the irradiation exposure time released by the 137Cs source according to the current exactor dose rate. For example, if the current dose rate equals 75.9 rad/min (0.759 Gy min−1 = 75.9 cGy m−1), the time of exposure in minutes is calculated as the desired dose/0.759. To irradiate the animals to a dose of 12 Gy TBI, the exposure time is ~15.81 min (15 min and 48 s).
  5. Disinfect the sample chamber in the gamma irradiator with 70% ethanol solution; place the absorbent mat and animals inside the sample chamber.
    NOTE: Sham-treated animals should be placed in the irradiation room but not exposed to gamma irradiation.
  6. Place the lid and close the chamber.
  7. Program the gamma irradiator to expose animals to 12 Gy TBI.
    1. Turn on the machine by turning the key into the START position.
    2. Enter the operator number using a numeric keyboard and confirm by pressing ENTER.
    3. Enter the PIN number using a numeric keyboard and confirm by pressing ENTER.
    4. Press 1 for options.
    5. Press 1 for timer settings.
    6. Press 1 for irradiation time.
    7. Enter the timer setting for the next cycle by using a numeric keyboard (hh:mm:ss format) and confirm by pressing ENTER.
    8. Confirm the settings once more by pressing ENTER.
    9. Go back to the home menu by pressing CLEAR 2x.
    10. Press START to initiate the exposure.
  8. Leave the room for the time of active exposure. The machine will stop automatically after the preset time elapses and will start beeping.
    CAUTION: Cesium-137 (137Cs) is a deadly hazardous substance (Health Hazard: H314). External exposure to large amounts of 137Cs can cause burns, acute radiation sickness, and even death. 
  9. Turn off the machine by turning the key into the STOP position.
  10. Open the sample chamber, take off the lid, transfer the animals back to the cage, and disinfect the sample chamber with 70% ethanol solution.
  11. Transfer the animals back to the conventional housing room and observe their condition post treatment.
  12. Monitor the animals' weight every day and euthanize them immediately (despite the planned time point) if any symptoms of altered well-being, distress, or weight loss exceeding 15% of the starting body weight are observed.
  13. Three hours prior to the planned euthanasia disinfect the abdominal area and, using a 28G insulin syringe, inject the mice with 100 µL of EdU stock solution (administered intraperitoneally).
  14. Collect proximal intestines at 0 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 168 h post irradiation.
    1. Perform CO2 euthanasia according to the standards of the home institution.
      CAUTION: Carbon dioxide is a hazardous substance (H280). Contains gas under pressure; may explode if heated. May displace oxygen and cause rapid suffocation.
    2. Then, dissect the proximal part of the small intestine, remove attached tissues, flush with cold DPBS using a 16 G straight feeding needle attached to a 10 mL syringe, fix with modified Bouin's fixative buffer using a 16 G straight feeding needle attached to a 10 mL syringe, cut open longitudinally, and roll the proximal intestines using the swiss-roll technique as described previously59.
  15. Place the tissues in a histological cassette and leave for 24-48 h at room temperature in a container with 10% buffered formalin. The volume of the formalin should be sufficient to fully cover the histological cassettes. When the incubation time elapses, using forceps, transfer the histological cassettes to the container filled with 70% ethanol. Then, proceed with tissue paraffin embedding.
    NOTE: Tissue paraffin embedding was performed by the Research Histology Core Laboratory at Stony Brook University. The procedure can be stopped here, and paraffin blocks can be stored at room temperature.
    ​CAUTION: Formalin is a potentially hazardous substance: Health Hazard (H350, H315, H317, H318, H370), and corrosive. Formalin may cause cancer, skin and eye irritation, damage to organs, and allergic skin reactions.

4. Histological analysis

  1. Cool down the histological cassettes containing the paraffin-embedded tissue specimens by placing them on ice for at least 1 h. Using a microtome, prepare 5 µm thick sections for staining. Every tissue should be sliced horizontally to cover the entire rolled specimen.
    1. After cutting the paraffin block, transfer the tissue sections to a water bath warmed up to 45 °C and place the sections on charged slides. Leave the slides on a rack overnight at room temperature to dry.
      NOTE: The procedure can be stopped here, and the slides can be stored at room temperature.
  2. Place the slides in a slide holder and bake in a 65 °C oven overnight. The slides can be baked for a shorter time, 1-2 h. However, this is not advisable in the case of freshly (less than 1 month old) cut slides.
    NOTE: Place the manual slide staining set under the chemical hood and perform the incubations with xylene, ethanol, and hematoxylin under the chemical hood.
  3. The next day, cool down the slides by placing them in a slide holder under the chemical hood for 10 min.
  4. Deparaffinize the tissues by placing the slide holder in a container filled with 100% xylene for 3 min. Repeat incubation using fresh 100% xylene.
    NOTE: From that moment, ensure the specimens are kept wet at all times.
    CAUTION: Xylene is a potentially hazardous substance: flammable (GHS02), causing acute toxicity (GHS07), and a Health hazard (H226 - H304 - H312 + H332 - H315 - H319 - H335 - H373 - H412). The potential hazardous effects are that it is fatal if swallowed or enters airways and can cause skin, eye, and respiratory irritation. Additionally, it may affect motor functions by causing drowsiness or dizziness and cause organ damage in case of prolonged or repeated exposure.
  5. Rehydrate sections in an ethanol gradient by placing the slide holder sequentially in containers filled with the following solutions: 100% ethanol, 2 min; 95% ethanol, 2 min; 70% ethanol, 2 min.
  6. Transfer the slide holder to the container filled with distilled water and rinse in running distilled water for 2 min.
  7. Place the slide holder in a container filled with hematoxylin solution, and stain for 5 min (under the chemical hood).
  8. Transfer the slide holder to the container filled with tap water and rinse in running tap water until the hematoxylin staining turns bluish, typically after 2 min.
  9. Transfer the slide holder to the container filled with 5% (w/v) lithium carbonate solution. Dip 10x.
    CAUTION: Lithium carbonate is a potentially hazardous substance: causing acute toxicity (GHS07), and a Health Hazard (H302 - H319). It is harmful if swallowed, harmful in contact with skin, and causes serious eye irritation.
  10. Transfer the slide holder to the container filled with distilled water and rinse in running distilled water for 2 min.
  11. Place the slide holder in a container filled with eosin and stain for 5 min.
  12. Transfer the slide holder to the container filled with 70% ethanol and rinse briefly (10 s).
  13. Dehydrate the sections by placing the slide holder sequentially in the containers filled with ethanol gradient solutions: 95% ethanol, 5 dips; 100% ethanol, 5 dips.
  14. Clear the slides by placing the slide holder in the container filled with 100% xylene for 3 min. Repeat incubation using fresh 100% xylene.
  15. Take the slide out of the rack, dry the area around the tissues using a paper towel, and depending on the size of the specimen, add one drop or two of xylene-based mounting medium on the surface of the specimen. Mount by gently placing a coverslip on top of the glass slide (be careful not to leave any air bubbles). Press gently if needed. The whole surface of the glass should be covered and sealed.
  16. Dry the slides by leaving them under the chemical hood overnight. Typically, the mounting medium solidifies in less than 24 h. To ensure the slides are dry, a sample slide can be made. Using an empty slide, place a drop of the mounting medium and leave under the chemical hood overnight. The next day, touch the drop and check if it is solidified.
  17. When the slides dry out, analyze the histology of the tissues using a light microscope or a more advanced microscope with a bright field channel.
    1. Place a microscope slide on the stage, move until the sample is in the center of the field of view, adjust the focus using the focus knob, and use a 10x and/or 20x magnification objective lens to analyze the histology in comparison to control tissues (sham irradiated).
    2. If the microscope is equipped with a camera, take images as well.
      ​NOTE: The procedure can be stopped here; the slides can be stored at room temperature and analyzed later. Do not keep the slides longer than 30 days as the staining slowly fades away.

5. Immunofluorescence staining

  1. Cool down the histological cassettes containing paraffin-embedded tissue specimens by placing them on ice for at least 1 h. Using a microtome, prepare 5 µm thick sections for staining. Every tissue should be sliced horizontally to cover the entire rolled specimen.
    1. After cutting the paraffin block, transfer the tissue sections to a water bath warmed to 45 °C and place the sections on charged slides. Leave the slides on a rack overnight at room temperature to dry.
      NOTE: The procedure can be stopped here, and the slides can be stored at room temperature.
  2. Place the slides in a slide holder and bake in a 65 °C oven overnight. The slides can be baked for a shorter time, 1-2 h. However, this is not advisable in the case of freshly (less than 1 month ago) cut slides.
    NOTE: Place the manual slide staining set under the chemical hood and perform the incubations with xylene, ethanol, and H2O2/methanol under the chemical hood.
  3. The next day, cool down the slides by placing them in a slide holder under the chemical hood for 10 min.
  4. Deparaffinize the tissues by placing the slide holder in a container filled with 100% xylene for 3 min. Repeat incubation using fresh 100% xylene.
    NOTE: From that moment, ensure the specimens are kept wet at all times.
  5. Quench endogenous peroxidase by incubating the slides for 30 min in a container filled with 2% hydrogen peroxide solution in methanol under the chemical hood.
    CAUTION: Methyl alcohol is a potentially hazardous substance: Health Hazard (H225-H301, H311, H331-H370), flammable (GHS02), and causes acute toxicity (oral, dermal, inhalation) (GHS08). Hydrogen peroxide is a potentially hazardous substance: corrosive (GHS05) and causes acute toxicity (GHS07).
  6. Rehydrate the sections in an ethanol gradient by placing the slide holder sequentially in containers filled with the following solutions: 100% ethanol, 3 min; 95% ethanol, 3 min; 70% ethanol, 3 min.
  7. Transfer the slide holder to a container filled with distilled water and rinse in running distilled water for 2 min.
  8. To retrieve the antigens, transfer the slide holder to a container with 250 mL of citrate buffer solution (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) and cook at 110 °C for 10 min using a decloaking chamber.
  9. Transfer the whole container to the cold room and allow gradual cooling down over the course of 30 min.
  10. After 30 min, replace the citrate buffer solution with distilled water and rinse in running distilled water until all floating paraffin pieces are removed.
  11. Take the slides out of the holder (one at a time), tap on a paper towel, and carefully dry the area around the specimen with a paper towel. Draw a closed shape around the specimen using a pen that provides a hydrophobic barrier (PAP pen). Place in a humidified chamber.
  12. Block with 5% bovine serum albumin (BSA) in TBS-Tween by adding 200 µL of the solution onto the surface of a specimen. Ensure there is no leakage. Incubate at 37 °C in a humidified chamber for 30 min.
  13. Remove the blocking solution by tapping the glass slide on a paper towel. Place back in a humidified chamber. Add 100 µL of the appropriate concentration of the primary antibodies resuspended in a blocking buffer and incubate at 4 °C with gentle rocking overnight. For BMI1/EYFP, use chicken anti-GFP (dilution 1:500); for Ki-67, use rabbit anti-Ki-67 (dilution 1:200).
  14. Remove the antibody solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to the container filled with TBS-Tween. Wash the slides with shaking for 5 min. Repeat 3x. Each time, use a fresh portion of TBS-Tween buffer.
  15. Take the slides out of the holder (one at a time), remove excess washing solution by tapping the glass slide on a paper towel, and place in a humidified chamber. Add 100 µL of the appropriate concentration of secondary antibodies resuspended in a blocking buffer, and incubate at 37 °C for 30 min. Secondary antibodies should be conjugated with a fluorophore. For BMI1/EYFP, use donkey anti-chicken Alexa Fluor 647 (dilution 1:500); for Ki-67, use goat anti-rabbit Alexa Fluor 488 (dilution 1:500).
  16. Remove the antibody solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to the container filled with TBS-Tween. Wash the slides with shaking for 5 min. Repeat 3x. Each time, use a fresh portion of TBS-Tween buffer.
  17. Take the slides out of the holder (one at a time), remove excess washing solution by tapping the glass slide on a paper towel, and place in a humidified chamber. Add 100 µL of the EdU staining solution prepared according to the manufacturer's instructions and using Alexa Fluor 555 fluorophore.
  18. Remove the EdU solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to the container filled with TBS-Tween. Wash the slides with shaking for 5 min. Repeat 2x. Each time, use a fresh portion of TBS-Tween buffer.
  19. Take the slides out of the holder (one at a time); remove excess washing solution by tapping the glass slide on a paper towel and place in a humidified chamber. Perform Hoechst 33258 counterstaining by adding 100 µL of the Hoechst 33258 solution (dilution 1:1000 in TBS-Tween) onto the surface of the specimen. Incubate in the dark at room temperature for 5 min.
  20. Remove the Hoechst 33258 solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to a container filled with TBS-Tween. Wash the slides with shaking for 5 min.
  21. Take the slide out of the slide holder, dry the area around the tissues using a paper towel, and depending on the size of the specimen, add one drop or two of aqua-based mounting medium onto the surface of the specimen. Mount by gently placing a coverslip on top of the glass slide (be careful not to leave any air bubbles). Press gently if needed. The whole surface of the glass should be covered and sealed.
  22. Dry the slides by leaving them in the dark at room temperature overnight. Typically, the mounting media solidifies in less than 24 h. To ensure the slides are dry, a sample slide can be made. Use an empty slide; place a drop of the mounting medium and leave under the chemical hood overnight. The next day, touch the drop and check if it is solidified.
  23. When the slides dry out, stained tissues can be analyzed using a fluorescence microscope equipped with emission wavelength filters allowing the visualization of Ki-67, EdU, and BMI1/EYFP staining (535 nm, 646 nm, and 700 nm, respectively).
  24. Place a microscope slide on the stage, move until the sample is in the center of the field of view, adjust the focus using the focus knob, and use the 10x and/or 20x magnification objective lens to analyze the staining in comparison to control tissues (sham irradiated). If the microscope is equipped with a camera, images can be taken as well. Take images for each channel separately and merge later.
    ​NOTE: The procedure can be stopped here; the slides can be stored at 4 °C and analyzed later. Do not keep the slides longer than 14 days as the staining slowly fades away.

6. TUNEL staining

  1. Cool down the histological cassettes containing paraffin-embedded tissue specimens by placing them on ice for at least 1 h. Using a microtome, prepare 5 µm thick sections for staining. Every tissue should be sliced horizontally to cover the entire rolled specimen.
    1. After cutting the paraffin block, transfer the tissue sections to a water bath warmed up to 45 °C and place the sections on charged slides. Leave the slides on a rack overnight at room temperature to dry.
      NOTE: The procedure can be stopped here, and the slides can be stored at room temperature.
  2. Place the slides in a slide holder and bake in a 65 °C oven overnight.
    NOTE: The slides can be baked for a shorter time, 1-2 hr. However, it is not advisable in the case of freshly (less than 1 month ago) cut slides. Place the manual slide-staining set under the chemical hood and perform the incubations with xylene and ethanol under the chemical hood.
  3. The next day, cool down the slides by placing them in a slide holder under the chemical hood for 10 min.
  4. Deparaffinize the tissues by placing the slides holder in a container filled with 100% xylene for 3 min. Repeat incubation using fresh 100% xylene.
    NOTE: From this point, ensure the specimens are kept wet at all times.
  5. Rehydrate the sections in an ethanol gradient by placing the slides holder sequentially in containers filled with the following solutions: 100% ethanol, 3 min; 95% ethanol, 3 min; 90% ethanol, 3 min; 80% ethanol, 3 min; 70% ethanol, 3 min.
  6. Transfer the slide holder to a container filled with distilled water and rinse in running distilled water for 1 min.
  7. Take the slides out of the holder (one at a time), tap on a paper towel, and carefully dry the area around the specimen with a paper towel. Draw a closed shape around the specimen using a pen that provides a hydrophobic barrier (PAP pen). Place in a humidified chamber.
  8. Incubate with DNase-free proteinase K. Add 100 µL of the DNase-free proteinase K solution (dilution 1:25 in DPBS) onto the surface of the specimen within the hydrophobic barrier. Incubate at room temperature in a humidified chamber for 15 min.
    CAUTION: Proteinase K is a potentially hazardous substance: Health Hazard (H315 - H319 - H334).
  9. Remove the proteinase K solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to the container filled with DPBS. Wash the slides with shaking for 5 min. Repeat 2x. Each time, use a fresh portion of DPBS.
  10. Prepare the TUNEL reaction mixture: Mix label solution (1x) with enzyme solution (10x). Pipette well.
  11. Take the slides out of the holder (one at a time); remove excess washing solution by tapping the glass slide on a paper towel and place in a humidified chamber. Add 50 μL of TUNEL reaction mixture onto the surface of the specimen and incubate in the dark at 37 °C in a humidified chamber for 60 min. For a negative control, use label solution only (without TdT).
  12. Remove the TUNEL solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to a container filled with DPBS. Wash the slides with shaking for 5 min. Repeat 2x. Each time, use a fresh portion of DPBS.
  13. Take the slides out of the holder (one at a time); remove excess washing solution by tapping the glass slide on a paper towel and place in a humidified chamber. Perform Hoechst 33258 counterstaining by adding 100 µL of the Hoechst 33258 solution (dilution 1:1000 in TBS-Tween) onto the surface of the specimen. Incubate in the dark at room temperature for 5 min.
  14. Remove Hoechst 33258 solution by tapping the glass slide on a paper towel; place the slides in a slide holder and transfer to the container filled with DPBS. Wash the slides with shaking for 5 min.
  15. Take the slide out of the slide holder; dry the area around the tissues using a paper towel, and depending on the size of the specimen, add one drop or two of aqua-based mounting medium onto the surface of the specimen. Mount by gently placing a coverslip on top of the glass slide (be careful not to leave any air bubbles). Press gently if needed. The whole surface of the glass should be covered and sealed.
  16. Dry the slides by leaving them in the dark at room temperature overnight. Typically, the mounting medium solidifies in less than 24 h. To ensure the slides are dry, a sample slide can be made. Place a drop of the mounting medium and leave in the dark at room temperature overnight. The next day, touch the drop and check if it is solidified.
  17. When the slides dry out, stained tissues can be analyzed using a fluorescence microscope equipped with emission wavelength filters allowing the visualization of TUNEL staining (535 nm).
  18. Place a microscope slide on the stage, move until the sample is in the center of the field of view, adjust the focus using the focus knob, and use a 10x and/or 20x magnification objective lens to analyze the staining in comparison to control tissues (sham irradiated). If the microscope is equipped with a camera, images can be taken as well. Take images for each channel separately and merge later.
    NOTE: The procedure can be stopped here; slides can be stored at 4 °C and analyzed later. Do not keep the slides longer than 14 days as the staining slowly fades away.

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Representative Results

The use of 12 Gy total-body irradiation (TBI) in combination with murine genetic lineage tracing allows for a thorough analysis of the consequences of radiation injury in the gut. To start, Bmi1-CreER;Rosa26eYFP mice received a single tamoxifen injection, which induces enhanced yellow fluorescent protein (EYFP) expression within a Bmi1+ reserve stem cell population. Two days subsequent to the tamoxifen injection, the mice underwent irradiation or sham irradiation. Three hours before euthanasia, the mice were injected with EdU. Following euthanasia, small intestine specimens were collected for analysis at 0 h, 3 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 7 days post irradiation. Representative hematoxylin and eosin (H&E) images (Figure 1) from the aforementioned time course illustrate the intestinal epithelium response to injury. The 0 h time point is illustrative of homeostatic intestinal epithelium (Figure 1); this is followed by the apoptotic phase-characterized by a loss of cells within crypt compartments between 3 h and 48 h (Figure 1). Then follows the regenerative phase, where highly proliferative cells can be found populating the crypts between 72 h and 96 h (Figure 1). Finally, this leads to the normalization phase, here represented by the 7 day time point (Figure 1).

Figure 2 illustrates changes in the proliferative status of intestinal crypt cells assessed by immunofluorescent staining of EdU (marker of cells in S-phase) and Ki-67 (marker of cells in G1, S, and G2/M phases), while lineage tracing (EYFP+ cells) marks cells originating from Bmi1+ reserve stem cells. During homeostasis (Figure 2, 0 h sham), the Bmi1+-positive cells marked by the expression of EYFP are restricted to the +4-+6 position within crypts. During the apoptotic phase (24 h and 48 h), EYFP-positive cells decrease in number (Figure 2). The regenerative phase (72 h and 96 h) is characterized by rapidly proliferating Bmi1+-positive cells and their progenitors (Figure 2); by 7 days post-irradiation, further proliferation and migration have replenished the intestinal crypt cells and restored intestinal epithelium integrity (Figure 2).

In sham-irradiated mice (control), EdU and Ki-67 stain EYFP negative proliferative cells of the intestinal crypts, typical of normal intestinal homeostasis, extending from active stem cells through the transit-amplifying zone (Figure 2). Radiation damage causes a loss of highly proliferative cells during the apoptotic phase, as shown by the reduction of EdU and Ki-67 staining (Figure 2). The activation of reserve stem cells (in this example, EYFP marked Bmi1+ positive cells) and their proliferation result in an increase in EdU and Ki-67 positive cells clearly visible at 72 h and 96 h post injury, where crypts are comprised almost exclusively of cells co-stained with EYFP, EdU, and Ki-67. The observed levels of proliferation normalize 7 days post injury (Figure 2).

In order to illustrate apoptosis caused by radiation damage, TUNEL staining was performed, and representative images are included in Figure 3. During homeostasis (Figure 3), few cells are undergoing apoptosis, typical of the normally rapid intestinal epithelial turnover. A clear increase in TUNEL staining can be observed late in the apoptotic phase (24 h and 48 h) following irradiation (Figure 3). During the regenerative phase, TUNEL staining steadily decreases within the regenerating crypts and is again nearly absent when the crypts have normalized (Figure 3). The presented protocol describes a simple and approachable immunofluorescent staining method utilizing a straightforward and widely employed combination of labeling techniques (TUNEL, EdU, Ki-67, EYFP) that is able to provide insights into the behavior of intestinal cells following injury (here, irradiation). This approach can be employed to study the mechanisms regulating regenerative processes in the intestinal epithelium in specific circumstances. Employing this and similar approaches will illuminate alternate regenerative processes following different intestinal insults and, further, will allow the investigation of therapeutic strategies for the prevention of loss of barrier function.

Figure 1
Figure 1: Representative images of hematoxylin and eosin (H&E)-stained sections of small intestinal tissues following a time course after total body irradiation. Bmi1-CreER;Rosa26eYFP mice received one dose of tamoxifen 2 days before 12 Gy total-body irradiation or sham irradiation. Mice were sacrificed, and small intestine tissues were collected at time points indicated in the figure. All images were taken at 10x magnification. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Representative images of immunofluorescent staining of small intestine sections following irradiation. Bmi1-CreER;Rosa26eYFP mice received one dose of tamoxifen 2 days before 12 Gy total-body irradiation or sham irradiation. Mice were sacrificed, and small intestine tissues were collected at time points indicated in the figure. Tissue sections were stained with EdU to mark cells in S-phase (pink) and with Ki-67 to mark cells in G0, S, and G2/M phases (yellow). EYFP (green) marks Bmi1+ cells and their progenitors and demonstrates lineage tracing. The sections were further counterstained with DAPI (blue) to visualize nuclei. The data are shown as merged images at 10x magnification and insets of individual stains at 20x magnification (these originated from 10x images). Please click here to view a larger version of this figure.

Figure 3
Figure 3: Representative images of immunofluorescent TUNEL staining of small intestine sections upon irradiation. Bmi1-CreER;Rosa26eYFP mice received one dose of tamoxifen 2 days before 12 Gy total-body irradiation or sham irradiation. Mice were sacrificed, and small intestine tissues were collected at time points indicated in the figure. TUNEL (red) stains apoptotic cells. These images were counterstained with DAPI (blue) to visualize nuclei. The images shown were taken at 20x magnification. Please click here to view a larger version of this figure.

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Discussion

This protocol describes a robust and reproducible radiation injury model. It allows for the precise analysis of the changes in the intestinal epithelium over the course of 7 days post injury. Importantly, the selected time points reflect crucial stages of injury and are characterized by distinct alterations to the intestine (injury, apoptosis, regeneration, and normalization phases)60. This model of irradiation has been established and carefully assessed, demonstrating a suitable manifestation of injury to mimic that experienced by patients undergoing radiotherapy61. More than half of patients with colorectal cancer and gynecological cancers undergo abdominal radiotherapy40,62,63. In spite of advanced methods for targeting radiotherapy, patients experience irradiation of healthy intestinal tissue, which produces pathophysiology very similar to that observed with the total-body irradiation model presented here. Therefore, the radiation model described herein can potentially address the repercussions of accidental radiation exposure of patients and/or medical personnel and, thus, is vital for human health.

The Bmi1-CreER;Rosa26eYFP mice model carries tamoxifen-inducible Cre under the transcriptional control of the mouse Bmi1 (Bmi1 polycomb ring finger oncogene) promoter. Additionally, these mice carry an R26-stop-EYFP sequence, where the STOP codon is flanked by loxP sides followed by the Enhanced Yellow Fluorescent Protein gene (EYFP) inserted into the Gt(ROSA)26Sor locus. The expression of EYFP is blocked by an upstream loxP-flanked STOP sequence but can be activated by tamoxifen-induced, Cre-mediated targeted deletions, allowing lineage tracing of Bmi1-expressing cells, a reserve stem cell-specific subpopulation of crypt cells. The radiation protocol in combination with the Bmi1-CreER;Rosa26eYFP mice model has been employed previously to study the role of transcription factors in regulating intestinal regeneration18,56. The representative results presented in Figure 1 to Figure 3 show results akin to those presented in recent publications18,56.

Of note, Bmi1-CreER;Rosa26eYFP mice are only one of multiple transgenic animal models employed to study intestinal regeneration. Other murine lineage tracing models have been combined with radiation to demonstrate the roles of specific cells and pathways in the regenerative capacity of the intestinal epithelium18,26,32,33,34,35,36,37,56,64,65,66,67. These models are designed to investigate the role of different populations of reserved stem cells, explore the precursors of secretory and enterocyte lineages, and study the function of specific genes within each subpopulation of the intestinal epithelial cells. Combined results from these investigations will help to expand the understanding of the regenerative capability of the intestinal epithelium.

Importantly, with minor modifications, this technique should allow for the investigation of the relationships between microbiota and different epithelial, stromal, and immune cell populations upon radiation injury. Introducing distinct lineage-tracing animal models will allow the study of real-time behavior and the interaction of various subpopulations of cells post injury. Alternatively, the staining of tissues could be replaced by RNA-sequencing, single-cell RNA-sequencing, fluorescence-activated cell sorting (FACS), or proteomic analysis to glean more in-depth knowledge about the role of specific cell lineages during intestinal regeneration following injury68,69. This injury model could be utilized to test new treatment modalities intended to minimize the side effects of irradiation, shorten recovery time, and improve the quality of patient care70. Although the radiation model described here can serve as a useful tool to study the regeneration of the intestinal epithelium, it also has several limitations. The total-body radiation dose invariably results in subsequent hematopoietic syndrome, which hinders the investigation of intestinal regeneration. This can be averted by bone marrow transplantation. Further, the modification to an abdominal-only injury protocol may potentially ameliorate some of the side effects of the irradiation71. However, this model requires additional steps that may affect gut response to the injury (e.g., anesthesia). Incidentally, abdominal radiation produces radiation-induced gastrointestinal syndrome (RIGS) without hematopoietic syndrome, differing from whole-body irradiation (WBI), in that WBI produces similar GI symptomology but includes partial loss of hematopoietic function following injury71,72.

Of note, genetically engineered animal models such as those described in this protocol are sophisticated and allow for the lineage tracing of specific cells. However, these models, though very beneficial, require additional effort to maintain. Transgenic animal models that allow for conditional gene deletions are difficult to generate, and their timely deletion upon injury may not be easily accomplished. Moreover, most transgenic models allow only for a conditional deletion or inactivation of a gene of interest but are not inducible and thus, are incapable of restoring gene function and may impede the studies. Furthermore, transgenic animal models usually require treatment with additional chemicals (e.g., tamoxifen, doxycycline) to achieve inactivation or overexpression of the gene of interest. Treatments with these substances may, to some degree, alter the response of the intestinal epithelium or add to the severity of the injury 73,74,75,76. In addition, the induction of lineage tracing by tamoxifen can be introduced closer to the time of radiation injury to improve the labeling of the cells involved in regeneration (e.g., 6-24 h before injury). Thus, it is possible to substitute a transgenic model with wild-type mice and a combination of IF staining (e.g., Ki-67, PCNA, EdU, TUNEL, Caspase 3). This model allows for the analysis of the regenerative capacity of the intestine, although it does not permit the study of a specific subpopulation of the cells.

The protocol described in this manuscript has several crucial steps. It is of utmost importance to ensure that healthy mice are used for experimentation, as they will be exposed to radiation and treatment with chemicals. If experiments with different mice groups are performed days, weeks, or months apart, it is good practice to adhere to the same timeline, e.g., tamoxifen injections at the same time of the day. The irradiation process should be done promptly, and mice should be returned to their original cages straight away. In addition, providing softened food and plenty of water can help to ameliorate some of the adverse effects of radiation. The swiss-roll technique is suggested to allow for comprehensive analysis of the intestinal tissue59. This technique is not trivial, and prior tryouts should be done to ensure that impeccable quality of the tissue for staining is achieved. All solutions for mice experiments and staining should be prepared freshly and stored under appropriate conditions. When working with antibodies, it is important to test slides using positive and negative controls and, if possible, use antibodies with the same catalog number and lot number. Immunofluorescence slides should be stored at 4 °C and imaged within 1 week of preparation. Prolonged storage, even at −20 °C, may result in the loss of signal. H&E slides can be stored at room temperature as there is no concern of loss of fluorophore stability.

In summary, the lineage-tracing radiation injury model presented here is a reproducible and relevant model to track intestinal cell fate following insult and can be combined with diverse murine models and molecular techniques to improve the understanding of the pathophysiology of the intestinal epithelium. Insight into the molecular and cellular mechanisms governing intestinal epithelium regeneration could lead to the development of new treatments and beneficial therapeutic interventions.

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Disclosures

The authors have no conflicts of interest.

Acknowledgments

The authors wish to acknowledge the Stony Brook Cancer Center Histology Research Core for expert assistance with tissue specimen preparation and the Division of Laboratory Animal Resources at Stony Brook University for assistance with animal care and handling. This work was supported by grants from the National Institutes of Health DK124342 awarded to Agnieszka B. Bialkowska and DK052230 to Dr. Vincent W. Yang.

Materials

Name Company Catalog Number Comments
1 mL syringe BD 309659 -
16G Reusable Small Animal Feeding Needles: Straight VWR 20068-630 -
27G x 1/2" needle BD 305109 -
28G x 1/2" Monoject 1mL insulin syringe Covidien 1188128012 -
5-Ethynyl-2′-deoxyuridine (EdU) Santa Cruz Biotechnology sc284628A 10 mg/mL in sterile DMSO:water (1:4 v/v), aliquot and store in -20°C
Azer Scientific 10% Neutral Buffered Formalin Fisher Scientific 22-026-213 -
B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J The Jackson Laboratory Strain #:006148
B6;129-Bmi1tm1(cre/ERT)Mrc/J The Jackson Laboratory Strain #:010531
Bovine Serum Albumin Fraction V, heat shock Millipore-Sigma 3116956001
Chicken anti-GFP Aves GFP-1020
Click-IT plus EdU Alexa Fluor 555 imaging kit, Invitrogen Thermo Fisher Scientific C10638 -
Corn oil Millipore-Sigma C8267 -
Decloaking Chamber Biocare Medical DC2012 -
Dimethyl sulfoxide (DMSO) Fisher BioReagents BP231-100 light sensitive
DNase-free proteinase K Invitrogen C10618H diluted 25x in DPBS
Donkey anti-chicken AF647 Jackson ImmunoResearch 703-605-155
DPBS Fisher Scientific 21-031-CV -
Eosin Fisher Scientific S176
Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10X, 20X Nikon
Fluoromount Aqueous Mounting Medium Millipore-Sigma F4680-25ML
Gamma Cell 40 Exactor Best Theratronics Ltd. - 0.759 Gy min-1
Goat anti-rabbit AF488 Jackson ImmunoResearch 111-545-144
Hematoxylin Solution, Gill No. 3 Millipore-Sigma GHS332
HM 325 Rotary Microtome from Thermo Scientific Fisher Scientific 23-900-668
Hoechst 33258, Pentahydrate (bis-Benzimide) Thermo Fisher Scientific H3569 dilution 1:1000
Hydrogen Peroxide Solution, ACS, 29-32%, Spectrum Chemical Fisher Scientific 18-603-252 -
In Situ Cell Death Detection Kit, Fluorescein (Roche) Millipore-Sigma 11684795910
Liquid Blocker Super PAP PEN, Mini Fisher Scientific DAI-PAP-S-M
Lithium Carbonate (Powder/Certified ACS), Fisher Chemical Fisher Scientific L119-500 0.5g/1L dH2O
Luer-Lok Syringe sterile, single use, 10 mL VWR 89215-218 -
Methanol VWR BDH1135-4LP
Pharmco Products Ethyl alcohol, 200 PROOF Fisher Scientific NC1675398 -
Pharmco-Aaper 281000ACSCSLT Acetic Acid ACS Grade Capitol Scientific AAP-281000ACSCSLT -
Rabbit anti-Ki67 BioCare Medical CRM325
Richard-Allan Scientific Cytoseal XYL Mounting Medium Fisher Scientific 22-050-262
Scientific Industries Incubator-Genie for baking slides at 65 degree Fisher Scientific 50-728-103
Sodium Citrate Dihydrate Fisher Scientific S279-500
Stainless Steel Dissecting Kit VWR 25640-002
Superfrost Plus micro slides [size: 25 x 75 x 1 mm] VWR  48311-703
Tamoxifen Millipore-Sigma T5648 30 mg/mL in sterile corn oil, preferably fresh or short-sterm storage in -20°C, light sensitive
Tissue-Tek 24-Slide Holders with Detachable Handle Sakura 4465
Tissue-Tek Accu-Edge Low Profile Blades Sakura 4689
Tissue-Tek Manual Slide Staining Set Sakura 4451
Tissue-Tek Staining Dish, Green with Lid Sakura 4456
Tissue-Tek Staining Dish, White with Lid Sakura 4457
Tween 20 Millipore-Sigma P7949
Unisette Processing Cassettes VWR 87002-292 -
VWR Micro Cover Glasses VWR 48393-081
Xylene Fisher Scientific X5P-1GAL

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Intestinal Epithelial Regeneration Ionizing Irradiation Gamma Irradiation Colorectal Cancer Treatment Gastrointestinal Tract Cell Activation Cell Differentiation Cell Migration Radiation Injury Model Intestinal Cell Fate Regenerative Capacity Lineage-tracing Animal Models Microbiota Epithelial Cell Population Stromal Cell Population Immune Cell Population Tamoxifen Powder Corn Oil EdU Powder DMSO Tissue Collection
Intestinal Epithelial Regeneration in Response to Ionizing Irradiation
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Orzechowska-Licari, E. J., LaComb,… More

Orzechowska-Licari, E. J., LaComb, J. F., Giarrizzo, M., Yang, V. W., Bialkowska, A. B. Intestinal Epithelial Regeneration in Response to Ionizing Irradiation. J. Vis. Exp. (185), e64028, doi:10.3791/64028 (2022).

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