Radiation enteropathy and leucocyte-endothelial cell reactions in a refined small bowel model

Abstract

Background: Leucocyte recruitment and inflammation are key features of high dose radiation-induced tissue injury. The inflammatory response in the gut may be more pronounced following radiotherapy due to its high bacterial load in comparison to the response in other organs. We designed a model to enable us to study the effects of radiation on leucocyte-endothelium interactions and on intestinal microflora in the murine ileum. This model enables us to study specifically the local effects of radiation therapy.

Method: A midline laparotomy was performed in male C57/Bl6 mice and a five-centimetre segment of ileum is irradiated using the chamber. Leucocyte responses (rolling and adhesion) were then analysed in ileal venules 2 - 48 hours after high dose irradiation, made possible by an inverted approach using intravital fluorescence microscopy. Furthermore, intestinal microflora, myeloperoxidase (MPO) and cell histology were analysed.

Results: The highest and most reproducible increase in leucocyte rolling was exhibited 2 hours after high dose irradiation whereas leucocyte adhesion was greatest after 16 hours. Radiation reduced the intestinal microflora count compared to sham animals with a significant decrease in the aerobic count after 2 hours of radiation. Further, the total aerobic counts, Enterobacteriaceae and Lactobacillus decreased significantly after 16 hours. In the radiation groups, the bacterial count showed a progressive increase from 2 to 24 hours after radiation.

Conclusion: This study presents a refinement of a previous method of examining mechanisms of radiation enteropathy, and a new approach at investigating radiation induced leucocyte responses in the ileal microcirculation. Radiation induced maximum leucocyte rolling at 2 hours and adhesion peaked at 16 hours. It also reduces the microflora count, which then starts to increase steadily afterwards. This model may be instrumental in developing strategies against pathological recruitment of leucocytes and changes in intestinal microflora in the small bowel after radiotherapy.

Figure 1

The chamber for segmental intestinal radiation exposure.

Figure 2

Venular leucocyte rolling in the mouse ileum at different time points after radiation. Data represents mean ± SEM.

Figure 3

Venular leucocyte adhesion in the mouse ileum at different time points after radiation. Data represents mean ± SEM.

Figure 4

A Cross section of intestinal wall 2 hrs after irradiation. No marked differences in the number of inflammatory cell types compared to the controls. B Cross section of intestinal wall 48 hrs after irradiation. A vast increase of goblet and apoptotic cells was seen in the whole length of the epithelium and crypts. There was a reduction in lymph vessel ectasia, oedema and in the number of inflammatory cells present compared to earlier time points.

Figure 5

Ileum bacterial microflora in sham and 2 hours after radiation groups. * denotes p < 0.05 compared to sham group.

Figure 6

Ileum bacterial microflora in sham and 16 hours after radiation groups. * denotes p < 0.05 compared to sham group.

Figure 7

Ileum bacterial microflora in sham and 24 hours after radiation groups. No significant difference between the experimental groups.

Figure 8

Ileum bacterial microflora in the radiated groups at different time points. * denotes p < 0.05 compared to 24 hours radiated group.