Name: intestinal epithelial regeneration in response to ionizing irradiation
Uploaded: 2022-07-27T14:05:00
Description: Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

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.

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-<em...

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 ...

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+,&#160;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&#39;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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intestinal epithelial regeneration in response to ionizing irradiation

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 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&#160;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 hour...

Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

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 ...

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