The frequent use of radiotherapy for abdominal and pelvic malignancies results in an increased risk of radiation enteritis [14
]. The dose of radiation that can be applied in clinical practice is usually limited by the need to restrict the number and severity of side effects in normal tissues surrounding a tumour, which are unavoidably exposed to radiation [8
]. Intestinal radiation toxicity (radiation enteropathy) is characterised by mucosal barrier breakdown and inflammation, followed by development of progressive vascular sclerosis and intestinal wall fibrosis. The process is accompanied by sustained over expression of inflammatory and fibrogenic cytokines [15
An early inflammatory response, beginning a few hours after irradiation, characterised by leucocyte infiltration into the irradiated organs is regarded as one of the main determinants of radiation-induced organ damage [17
]. The development of an inflammatory response involves sequential leucocyte-endothelial cell interactions. Different families of cell adhesion molecules have been shown to participate in the process of leucocyte recruitment [19
]. There are three major families of adhesion molecules involved in the leucocyte recruitment process, the selectins, the integrins and the immunoglobulin supergene families [20
The present study has concentrated on the acute effects of radiation injury on leucocyte rolling and adhesion at specific time points after radiation. We found that radiation evoked a marked time dependent leucocyte response with a significant increase in both leucocyte rolling and firm adhesion over time. Leucocyte rolling peaked 2 hours after radiation whereas leucocyte adhesion was highest after 16 hours showing a marked response. Interestingly, both the leucocyte rolling and adhesion responses to radiation were back to baseline 48 hours after radiation. An intravital microscopic study of radiation-induced leucocyte-endothelial cell interaction using abdominal radiation and a dose of 20 Gy revealed an increased leucocyte rolling in mesenteric venules 2 hours after radiation, with a marked increase in leucocyte adhesion and emigration noted at 6 hours [18
]. In another study of radiation-induced inflammatory damage, abdominal irradiation was administered using 4 and 10 Gy respectively [8
]. Here an increase in leucocyte rolling was observed 2 hours after radiation, which then returned to basal levels at 6 and 24 hours respectively. An increase in leucocyte adhesion was also observed 2 hours after irradiation, which was then sustained during the 24 hour observation period [8
]. In our study we showed the maximum effect on rolling after 2 hours and adhesion after 16 hours and the return to basal levels 48 hours after radiation. We used a single high dose radiation of 19 Gy directly to an exteriorised segment of ileum. This dose was chosen because it has been shown to give a good correlation or dose response relationship of histopathological changes (e.g. mucosal ulceration, vascular sclerosis) to incidence of clinical complications and cellular evidence of injury [1
]. When comparing our results (ileal venule measurements) to those from other tissue, namely from the pial venules of cerebral microvasculature of the rat after 20 Gy irradiation [21
], we find that the results follow a similar time course. Assuming that the radiation dose distribution is similar in all experiments mentioned above, the differences in peak times for leucocyte rolling and adhesion may probably be due to differences in radiation dose/duration, the extent of trauma, the effect of anaesthesia, the mode and duration of experiments.
Endogenous bacterial flora produces nutrients (e.g. short-chain fatty acids) for the mucosa; prevents overgrowth of potentially pathogenic micro-organisms; stimulates the immune system especially the gut-associated lymphoid tissue; helps eliminate toxins from the lumen and participates in intestinal regulation, motility and blood flow [22
]. Radiation on the other hand influences and alters the mucosal microflora, and this in combination with barrier dysfunction leads to a translocation of microbes through the mucosa into blood circulation [23
]. Our experiment shows that radiation affects the intestinal microflora. Two hours after radiation the aerobic, anaerobic, Enterobacteriaceae
counts were decreased and after 16 hours the aerobic, Enterobacteriaceae
counts were still decreased in the radiated groups compared to sham controls. Twenty-four hours after radiation there was no significant difference between the experimental groups. Comparing the results of the irradiated groups alone, one observes an increase in bacterial count over time after radiation. It seems that radiation decreases the bacterial count at early time points with no difference in total bacterial count at late time points. This total count does not reflect the difference in bacterial species within each group, and thus, further investigations are needed to study the imbalances that occur. One study has shown that microorganisms such as Escherichia, Proteus, Clostridium, normally absent in healthy animals, appear in the intestines of guinea pigs subjected to irradiation. At the same time lactobacilli and bifidobacteria sharply decrease in number [24
]. Bacterial overgrowth and intestinal pseudo-obstruction may succeed abdominal radiotherapy and the impaired motility emerges as a causal factor for gastrointestinal colonization with gram-negative bacilli. Abnormal motility and gram-negative bacilli in the gut may be essential in the pathogenesis of late radiation enteropathy [25
]. Changes in intestinal microflora therefore most probably affect the course of the development of radiation enteropathy. Acute intestinal symptoms during pelvic radiotherapy may not depend only on mucosal damage [26
]. Post-radiation gut structural damage occurs early and parallels functional changes of the intestinal mucosa, including increased epithelial permeability (shown both in vivo and ex vivo), activation of secretory pathways, decreased nutrient absorption, diarrhoea, and weight loss [27
]. The microfloral changes, which we have shown, could play an important role in the structural and functional intestinal changes after radiation, particularly in the presence of intestinal mucosal changes and increased intestinal permeability. Patients with carcinoma of the uterine cervix or endometrium receiving postoperative radiation therapy have a significant decrease in intestinal microflora after the first radiation exposure, whereas at the end of radiotherapy all bacteria have increased and reached basal values except Enterococcus faecium
1, lactobacilli and total anaerobes. In some patients an overgrowth of some Clostridium spp. (potential pathogens) associated with clinical symptoms, was observed. Patients receiving radiotherapy may thus benefit from the intake of oral bacteriotherapy [28
]. The importance of investigating the effects of radiation on the different bacterial species within the total count is therefore of significance for the modulation of treatment regimes.
The histological changes following radiation are both time and dose dependent [29
]. Soon after radiotherapy we observed an increase in inflammatory cell-infiltrate, apoptosis, mucin producing goblet cells and oedema, representing the morphological expression of an unspecific reactive process with a supposed protective function. Variations of these changes have been previously observed in the clinical situation. The vast increase in goblet cells that we observed may resemble that seen in necrotising enterocolitis. A resemblance to chronic idiopathic inflammatory bowel disease, eosinophilic colitis and microscopic colitis can also be seen if the mild crypt distortion or withering that occurs with radiation injury is confused with proper crypt architectural distortion of inflammatory disease. Isolated crypts due to nuclear regenerative changes may also mimic the microadenomas of familial adenomatosis polyposis [30
]. Histological changes in the pre-existing normal mucosa following preoperative radiotherapy need to be appreciated by the histopathologist if we are to avoid erroneous concurrent diagnosis [30
]. Furthermore, a correct assessment of the effects of new treatment regimes or prophylaxis is based on a sound histological judgment.
No differences MPO values could be seen between the controls and the radiated groups. This is probably because it is a crude method of measurement and thus may not be sensitive enough to detect early changes of inflammation.
This study therefore presents a refinement of previous methods of examining effects of radiation enteropathy, and a new approach at investigating radiation induced leucocyte responses in the ileal microcirculation. This new model may be instrumental in developing strategies against pathological recruitment of leucocytes and changes in intestinal microflora in the small bowel.