Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

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.

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-Cre^ER;Rosa26^eYFP 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.

Figure 2:. Representative images of immunofluorescent staining of small intestine sections following irradiation.

Bmi1-Cre^ER;Rosa26^eYFP 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).

Figure 3:. Representative images of immunofluorescent TUNEL staining of small intestine sections upon irradiation.

Bmi1-Cre^ER;Rosa26^eYFP 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.