A Fiber-Rich Diet and Radiation-Induced Injury in the Murine Intestinal Mucosa

Dietary fiber is considered a strong intestinal protector, but we do not know whether dietary fiber protects against the long-lasting mucosal damage caused by ionizing radiation. To evaluate whether a fiber-rich diet can ameliorate the long-lasting pathophysiological hallmarks of the irradiated mucosa, C57BL/6J mice on a fiber-rich bioprocessed oat bran diet or a fiber-free diet received 32 Gray in four fractions to the distal colorectum using a linear accelerator and continued on the diets for one, six or 18 weeks. We quantified degenerating crypts, crypt fission, cell proliferation, crypt survival, macrophage density and bacterial infiltration. Crypt loss through crypt degeneration only occurred in the irradiated mice. Initially, it was most frequent in the fiber-deprived group but declined to levels similar to the fiber-consuming group by 18 weeks. The fiber-consuming group had a fast response to irradiation, with crypt fission for growth or healing peaking already at one week post-irradiation, while crypt fission in the fiber-deprived group peaked at six weeks. A fiber-rich diet allowed for a more intense crypt cell proliferation, but the recovery of crypts was eventually lost by 18 weeks. Bacterial infiltration was a late phenomenon, evident in the fiber-deprived animals and intensified manyfold after irradiation. Bacterial infiltration also coincided with a specific pro-inflammatory serum cytokine profile. In contrast, mice on a fiber-rich diet were completely protected from irradiation-induced bacterial infiltration and exhibited a similar serum cytokine profile as sham-irradiated mice on a fiber-rich diet. Our findings provide ample evidence that dietary fiber consumption modifies the onset, timing and intensity of radiation-induced pathophysiological processes in the intestinal mucosa. However, we need more knowledge, not least from clinical studies, before this finding can be introduced to a new and refined clinical practice.

3D cyclorama for digital unrolling and visualisation of deformed tubes

Colonic crypts are tubular glands that multiply through a symmetric branching process called crypt fission. During the early stages of colorectal cancer, the normal fission process is disturbed, leading to asymmetrical branching or budding. The challenging shapes of the budding crypts make it difficult to prepare paraffin sections for conventional histology, resulting in colonic cross sections with crypts that are only partially visible. To study crypt budding in situ and in three dimensions (3D), we employ X-ray micro-computed tomography to image intact colons, and a new method we developed (3D cyclorama) to digitally unroll them. Here, we present, verify and validate our ‘3D cyclorama’ method that digitally unrolls deformed tubes of non-uniform thickness. It employs principles from electrostatics to reform the tube into a series of onion-like surfaces, which are mapped onto planar panoramic views. This enables the study of features extending over several layers of the tube’s depth, demonstrated here by two case studies: (i) microvilli in the human placenta and (ii) 3D-printed adhesive films for drug delivery. Our 3D cyclorama method can provide novel insights into a wide spectrum of applications where digital unrolling or flattening is necessary, including long bones, teeth roots and ancient scrolls.

How to Obtain a Mega-Intestine with Normal Morphology: In Silico Modelling of Postnatal Intestinal Growth in a Cd97-Transgenic Mouse

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Asymmetric crypt fission in colectomy specimens in patients with ulcerative colitis

AimsWe previously found colonic crypts with asymmetric fission bordering regenerating ulcers in ulcerative colitis (UC). The present objective was to assess the frequency of asymmetric crypt-fission in colectomy specimens from patients with long-lasting UC.MethodsH&E-stained sections from seven colectomies from patients with UC without dysplasia or carcinoma were investigated. Symmetric fission was characterised by branched colon crypts showing ≥2 identical crypts, whereas asymmetric fission exhibited branched colon crypt portraying ≥2 dissimilar crypts, differing in diameter, length and/or shape.ResultsThe number of crypts in fission in the 89 sections was 3586; of those, 2930 (81.7%) were asymmetric and the remaining 656 (18.3%), symmetric. Out of 927 vertically-cut crypts (in well-oriented sections), 912 (98.4%) were asymmetric, and the remaining 14 (1.6%), symmetric, and out 2660, cross-cut (transected) crypts in fission, 2018 (75.9%) were asymmetric and the remaining 642 (24.1%), symmetric.ConclusionCrypt fission is rarely found in the normal colon in adults. Symmetric crypt fission found in UC is possibly triggered by a compensatory homeostatic mechanism of crypt production in mucosal areas replaced by chronic inflammation. But asymmetric crypt fission is a pathological aberration that affects crypts in patients with a particular predisposition to develop mucosal dysplasia. It is suggested that this previously unattended histological parameter be included in the pathological descriptions of colectomy specimens from patients with UC.

Asymmetric crypt fission in sessile serrated lesions

ObjectiveSessile serrated lesions without dysplasia (SSL-ND) are epitomised by dilated crypts with epithelial serrations and architectural distortions portraying boot-shapes, L-shapes or inverted-T shapes. Recently, crypts in asymmetric fission were detected in SSL-ND. The purpose was to assess the frequency of crypts in asymmetric fission in a cohort of SSL-ND.MethodsThe frequency of crypts in fission was assessed in 60 SSL-ND, the distribution of cell proliferation in 48 SSL-ND and the expression of maspin, a tumour-suppressor protein, in 29 SSL-ND.ResultsOut of the 60 SSL-ND, 40 (66.7%) showed crypts in fission: 39 (65%) SSL-ND had crypts in asymmetric fission and one SSL-ND (1.7%), in symmetric fission (p<0.05). Of 1495 crypts recorded in the 60 SSL-ND, 73 (4.9%) were in asymmetric fission but only one (0.06%), in symmetric fission (p<0.05). Out of the 48 Ki67-immunostained SSL-ND,15 (31%) showed randomly distributed proliferating cell-domains. All 29 SSL-ND revealed maspin-upregulation (including crypts in asymmetric and symmetric fission). In contrast, the normal colon mucosa showed occasional single crypts in symmetric fission, proliferating cell-domains limited to the lower thirds of the crypts, absence of crypts in asymmetric fission and remained maspin negative.ConclusionsSSL-ND thrive with crypts in asymmetric fission displaying randomly distributed proliferating cell-domains and maspin-upregulation. These histo-biological aberrations disclose pathological cryptogenesis and suggest possibly unfolding somatic mutations in SSL-ND. The present findings may open new vistas on the parameters pertinent to the susceptibility of SSL-ND to develop dysplasia and carcinoma.

A diffusion-like process enables expansion of advantaged gene mutations in human colonic epithelium

Colorectal cancer is thought to arise when the mutational burden of the clonal population of stem cells within a colonic crypt exceeds a certain threshold. Therefore, quantification of the fixation and subsequent expansion of somatic mutations in histologically normal epithelium is key to understanding colorectal cancer initiation. Here, using immunohistochemistry, loss of the histone demethylase KDM6A in normal human colonic epithelium is visualised. Interpretation of the age-related behaviour of KDM6A-negative clones revealed significant competitive advantage in intra-crypt dynamics. Further, subsequent clonal expansion into multi-crypt patches was quantified to reveal a significant 5-fold increase in crypt fission rate. To accomodate the local accumulation of new crypts, the role of crypt fusion was considered. However, no compensatory increase in fusion rate was found. Instead, evidence for crypt diffusion is presented and proposed as a means of accommodating clonal expansions. The threshold fission rate at which diffusion fails to accommodate new crypts, and which may promote polyp growth, is defined.

Hyaluronic acid promotes Lgr5+ stem cell proliferation and crypt fission through TLR4 and PGE2 transactivation of EGFR

This study, in newborn mice, describes a novel molecular pathway regulating Lgr5+ epithelial stem cell proliferation and normal intestinal elongation, as assessed by crypt fission. In this pathway, endogenous extracellular hyaluronic acid binds to Toll-like receptor 4 on pericryptal macrophages releasing PGE2 which binds to epidermal growth factor receptor on Lgr5+ stem cells resulting in proliferation. Lgr5+ stem cell proliferation leads to crypt fission and intestinal elongation. The demonstration that normal growth requires microbial-independent Toll-like receptor activation is novel.

Intestinal stem cells promote crypt fission during postnatal growth of the small intestine

ObjectiveWnt-β-catenin signalling is essential for intestinal stem cells. Our aim was to investigate the relationship between intestinal stem cells and crypt fission which peaks during infancy.DesignDuodenal biopsies were obtained during endoscopy to assess the severity of reflux oesophagitis of 15 infants, children and teenagers, which would not affect the duodenum. Samples of small intestine were also obtained from rats 7–72 days of life. Crypt fission was assessed using microdissection of 100 whole crypts and recording the percentage of bifid crypts. Intestinal LGR5+ stem cells were identified by in situ hybridisation. Rats were treated with Dickkopf to block Wnt-β-catenin signalling.ResultsCrypt fission peaked during infancy before declining after 3–4 years in humans and after 21 days of life in rats. Occasional mitotic figures were seen in bifid crypts during early fission. Stem cells were elevated for a greater period during infancy and childhood in humans. Clustering of Paneth cells was present around the stem cells at the crypt base. Dickkopf reduced the number of stem cells and crypt fission to 45% and 29%, respectively, of control values, showing dependence of both crypt fission and Lgr5+ stem cells on Wnt signalling. However, Dickkopf did not decrease mitotic count per crypt, indicating a difference in signalling between stem cells and their progeny in the transit amplifying zone.ConclusionCrypt fission peaks during infancy and is dependent on intestinal stem cells. This is relatively hidden by ‘a cloak of invisibility’ due to the low proliferation of stem cells.