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.
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.
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.
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
2. Preparation of animals and materials
3. Total-body gamma irradiation (TBI) and tissue collection
4. Histological analysis
5. Immunofluorescence staining
6. TUNEL staining
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: 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: 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: 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.
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.
The authors have nothing to disclose.
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.
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 |