Acute changes in leukocyte populations following focal irradiation of the intestine.

Abstract

Radiotherapy is indicated for the treatment of half of all cancer patients. However, incidental irradiation of the bowel results in dose‐limiting side effects in the majority of patients receiving abdominal or pelvic radiation treatment. The major late stage complication in the irradiated bowel is connective tissue fibrosis. In the intestine, this process is likely driven by the immune response in the acute phase following focal irradiation. We recently developed a mouse model which involves surgically implanting a radiopaque marker onto the surface of the intestine in order to replicate highly focal clinical radiotherapy. Mice were then imaged with cone beam computed tomography to locate the marker, and irradiated with 18 Gy of 5x5 mm collimated x‐rays onto the marked intestine using the Small Animal Radiation Research Platform (SARRP). Irradiated mice exhibited 100% survival with minimal weight loss after irradiation when compared to mock‐irradiated controls. Irradiation significantly impaired crypt regeneration as assessed by a 5‐ethynyl‐2’‐deoxyuridine (EdU) cell proliferation assay one day post‐irradiation. Furthermore, a TdT dUTP nick end labeling (TUNEL) assay indicated a localized increase in crypt apoptosis in the irradiated area, suggesting the marked intestine was successfully irradiated. Beginning 3.5 days after irradiation, there was significant ablation of intestinal structure with very few proliferating cells in the irradiated area. The intestinal barrier was maintained in the acute phase by markedly hyperplastic crypts on the edges of the irradiated area. Furthermore, a severe innate immune response was observed in the irradiated intestine 3.5, 7, and 14 days post‐irradiation as evidenced by increased neutrophil and macrophage infiltration. This inflammatory process induced a systemic leukocytosis which was driven by an increase in neutrophils in the peripheral blood. Two months post‐irradiation, fibrosis was observed in the irradiated area by Masson’s trichrome stain. Our results suggest that a major inflammatory reaction in the acute phase precedes fibrosis.