In this study we have presented a new mucosal mapping technique for measuring markers of events in the colorectal mucosa and shown that it is simple to use, safe, and very reliable, and that patient compliance is good. These characteristics demonstrate the potential of the system as a practical clinical tool for evaluating inflammatory markers in many settings.
Compared with other methods,4,5,8,16,17
the present system has several advantages. In our experience it is technically simpler to perform and more comfortable for patients than rectal dialysis or rectal perfusion techniques.4,5
The size of the molecule to be evaluated is not critical, as it is in rectal dialysis. It does not require an endoscopic procedure, as in rectal perfusion and in the filter technique described by Hendel and colleagues,16
but it can be easily combined with endoscopy when required. The perfusion technique has been described as the golden standard for measurement of inflammatory mediators in the gut but its use is limited to research studies because of its complexity. Perfusion of the gut also dilutes the amounts of inflammatory substances, which means that low levels in non-inflammatory conditions and healthy controls are below the detection limit.18
Our new technique is very sensitive and permits detection of inflammatory cells even in normal mucosa by virtue of the extreme absorption capacity of the cellulose material used for the test procedure. Studies of the time needed for the cellulose material to absorb inflammatory substances from the mucosa indicated that after less than 20 minutes, equilibrium between the mucosa and cellulose was reached. For practical clinical purposes it seems quite acceptable to reduce the time of contact between the cellulose material and the mucosa to approximately 10 minutes. Studies in vitro of the highly absorptive cellulose patches demonstrated acceptable recovery of absorbed inflammatory substances with the extraction procedure used.
Preparation of the gut is essential in situations where the release of inflammatory substances to the gut lumen is measured as faecal contamination may influence the results.19–21
It has also been reported that a hyperosmolar solution may affect the mucosa if osmolarity is more than 1500 mosmol/l.22
The enema we used has an osmolarity of 1686 mosmol/l. However, preparation with an isotonic fluid and a hyperosmolar enema gave identical results in the present system and neither method induced any apparent inflammatory reaction in the rectal mucosa; these findings are in accordance with the results obtained with gut preparation prior to the perfusion technique.7
In this context it should be mentioned that repeated exposure of the rectal mucosa to a hyperosmolar milieu (2–3 enemas within 24 hours) induced an inflammatory reaction in some healthy controls (unpublished observations).
One advantage of the perfusion technique for assessing the inflammatory activity of the rectal mucosa is that results obtained by perfusion of a defined gut segment give an average value for mucosal inflammation.5,8
Patchiness of mucosal inflammation in UC has previously been emphasised.23,24
In an attempt to compensate for possible regional differences in the inflammatory status of the rectal mucosa, we performed three measurements at different rectal sites. The average intraindividual variation of the three rectal measurements was 38%, thus illustrating the expected variability in a mucosal inflammatory process. The difference in the mean of such measurements performed at different times was not significant. Thus reproducibility was quite acceptable with this process, which implies that longitudinal studies of the condition of the mucosa can be made in individual patients.
Another strength of the present technique is its ability to detect the activity of neutrophil and eosinophil granulocytes in apparently healthy rectal mucosa. With the intestinal perfusion technique we have not previously been able to define the normal activity of inflammatory cells in the gut mucosa.7,18,19
The advantage of a high sensitivity method was obvious in the present study as subclinical inflammation in the rectal mucosa was detected in patients with IBS. Although IBS is not regarded as an inflammatory disease, its inflammatory basis has been discussed, and non-specific mild inflammation in the colonic mucosa is often seen histologically in IBS patients.25–27
It has been proposed that such mucosal inflammatory findings might be explained by undiagnosed coeliac disease or postinfective IBS.27,28
However, such possible explanations are not relevant to our findings in IBS as only two of our IBS patients had a history of IBS secondary to a gastrointestinal infection which occurred more than three years before the present investigation. Furthermore, all of our IBS patients had normal duodenal biopsies and were negative for serological markers of coeliac disease.
Previous examinations of colonoscopic biopsy specimens from patients meeting the Rome criteria for a clinical diagnosis of IBS have given conflicting results. Chadwick and colleagues27
promoted the hypothesis that in at least a subset of patients with IBS low grade inflammation with increased numbers of neutrophils and mast cells might play a pathophysiological role. Others29
have reported, in this disease, increased numbers of mast cells in proximal colonic mucosa but no increase in these cells or neutrophils in the rectum. As overt colonic inflammation precludes a diagnosis of IBS, the need for more sensitive markers of low grade inflammation has been stressed.29
Counting single inflammatory cell subtypes per high power field has the potential to be more sensitive compared with our histological method (validated for UC to assess the degree of mucosal inflammation). Conventional histological examination of biopsies identifies the number of inflammatory cells but gives no information on the degree of granulocyte activation. Increased release of granular constituents is one sign of granulocyte activation. Our finding of increased mucosal amounts of neutrophil granular proteins (MPO and HNL) in spite of no apparent increase in neutrophils might therefore reflect low grade inflammation. This interpretation is supported by the finding that IBS patients have significantly increased amounts of faecal calprotectin, a neutrophil derived protein.30
Faecal protein amounts may originate from any part of an inflamed gut. We cannot fully exclude the fact that mediators absorbed in the rectum by the patches to some extent may originate from other sites of an inflamed colon. However, our preinvestigation preparation of patients and local application of the absorbent patches reduce such influences. The correlation between measured inflammatory mediators and endoscopic and histological findings in UC indicate that our technique reflects mainly a local inflammatory process.
The finding of subclinical rectal inflammatory activity in diet treated coeliac disease was unexpected but previous reports of proctitis in this disease deserve attention.31,32
Cellier et al
found that a gluten free diet induced regression but not normalisation of T cell activation in the rectal mucosa of patients with coeliac disease.32
Their report together with our present results suggests a non-gluten related increase in the inflammatory propensity of the gut mucosa in this disease. It is well known that gluten induces not only a lymphocytic response but also increased mucosal neutrophil infiltration.33
Patients with active UC showed, on average, more than 300-fold increase in the inflammatory activity of the rectal mucosa, as judged by recovered amounts of MPO. The involvement of eosinophils in this disease was also clear from the several-fold increase in the recovery of ECP, and our findings are in accordance with our previous report using a perfusion technique.7
Patients with inactive UC showed normalisation of eosinophil activity of the mucosa but neutrophil activity remained increased compared with that of healthy controls but of the same magnitude as observed in IBS and coeliac disease. The potential of this sensitive method for assessment of inflammatory activity was also illustrated by the significantly increased recovery of neutrophil granule constituents in our patients with CC. In a minority of these patients, mucosal recovery of ECP was highly increased in accordance with recently published data from a study using rectal perfusion.34
In summary, we have described a new principle for assessing biological events in the colonic mucosa and have applied it in studies of the rectal mucosa in health and in patients with various inflammatory and non-inflammatory bowel diseases. We conclude that this new technique is rapid, simple, safe, and highly sensitive, and produces reliable and reproducible data. The results obtained indicate that it might be suitable not only for practical clinical use but also for research studies of gut pathophysiology.