To best measure cell proliferation in samples from the intestine.

Arrangement of the intestinal cell lining, as it is, into distinct anatomically defined zones where proliferation is confined to the crypts, makes it an ideal tissue to study growth control mechanisms. While many methods have been used to quantify cell proliferation in the gut, several of them have severe limitations and others (although potentially better) have been misused and misinterpreted. Here, correct use and interpretation of labelling studies will be described as will a well established alternative method that provides equivalent results for one-sixth of the effort.

A simple device to rapidly prepare whole mounts of murine intestine.

Many mouse models of neoplasia and pre-neoplasia require the examination of whole mounts of the gastrointestinal tract. A simple device has been produced to facilitate the rapid preparation of mouse intestines for subsequent quantification of tumours and pre-neoplastic lesions such as aberrant crypt foci. The device greatly speeds up the production of whole mounts and also provides far more consistent and better-quality preparations.

The incidence of aberrant crypt foci and colonic carcinoma in dimethylhydrazine-treated rats varies in a site-specific manner and depends on tumor histology.

In an attempt to demonstrate the relationship between aberrant crypt foci (ACF) and subsequent colonic neoplasms, we investigated the distribution of ACF in the dimethylhydrazine (DMH) model of colonic carcinogenesis in the rat. DMH was given to male Wistar rats by s.c. injection in a dosage of 15 mg/kg body weight once a week for 19 weeks. As a result, eight poorly differentiated, mucin-secreting carcinomas, two well-differentiated tubular adenocarcinomas, and four adenomas developed in 35 rats autopsied at 24 weeks after the first injection of DMH. The location of each type of tumor was site specific. Poorly differentiated, mucin-secreting carcinomas of signet-ring type occurred only in the proximal colon; the mean location of these lesions was 17.6 +/- 3.8% (SE; range, 0-39%) of the length of the colon. Well-differentiated tubular adenocarcinomas and adenomas developed in the distal colon; the mean location of these lesions was 76.7% +/- 4.9 (SE; range, 60-90%) of the length of the colon. There was a mean number of 276 +/- 29 (SE) ACF per colon; these were present at between 40 and 90% of the colonic length, peaking at 70%. We conclude that ACF are marker lesions for colonic neoplasms, but only in the distal colon where tumors follow the adenoma-carcinoma sequence; this is not so for the proximal colon, where poorly differentiated, mucin-secreting carcinomas are found. These findings suggest that these latter tumors well may arise de novo and indicate that studies that attempt to correlate ACF with subsequent tumor formation must take cognizance, not only of the site, but also of the tumor type.

Leptin, cell proliferation and crypt fission in the gastrointestinal tract of intravenously fed rats.

Many peptides, hormones and growth factors have been implicated in the control of cell renewal in the gastrointestinal epithelium. Leptin is present in the stomach and salivary glands and leptin receptors are seen throughout the gut. Leptin can stimulate mitogen-activated protein kinase activity in vitro and short-term infusion has been reported to have a proliferative action on the colon in vivo, suggesting a biological link between obesity, physical activity and colon cancer. Food intake is one of the most important determinants of intestinal mucosal cell renewal, thus any direct effects of leptin on the gut may be hidden. This problem has been avoided experimentally by maintaining animals on total parenteral nutrition (TPN). Male Wistar rats were anaesthetized and cannulae were inserted into the jugular vein to deliver the TPN diet to which had been added 0, 0.5, 2.5, or 10 mg/kg of recombinant murine leptin. Orally fed rats were also studied. After 6 days of treatment, all animals were injected with vincristine and killed 2 h later. Tissue weight was recorded and crypt cell proliferation (arrested metaphases) and crypt fission were scored in 'microdissected' crypts. Leptin infusion led to a small decrease in body weight and in the weight of the caecum. Intestinal cell proliferation was significantly reduced by TPN when compared to the orally fed rats, but the addition of leptin had no effect on the small intestine or colon. Crypt fission was also significantly lowered in the TPN group. Fission was slightly but significantly increased in the proximal and mid-colon of the leptin-treated rats, but was decreased in the distal colon. Although leptin did not significantly alter cell proliferation, it had significant effects on the process of crypt fission in the colon, which varied according to the exact locality.

Cell proliferation in the small intestine and colon of intravenously fed rats: effects of urogastrone-epidermal growth factor.

There is marked intestinal hypoplasia in the intestine of intravenously fed (TPN) rats. Recombinant urogastrone-epidermal growth factor (URO-EGF) reversed these changes by significantly increasing the length of the intestinal crypts. Crypt diameter, however, was not affected to the same extent. Few differences in labelling indices were seen between the orally fed and TPN groups, however, this was the consequence of the concomitant changes in crypt population. The number of mitoses and labelled cells per crypt, and thus the crypt cell production rates, were significantly decreased in the TPN group when compared to the orally fed. URO-EGF significantly increased both proliferative indices and the number of dividing cells per crypt. Crypt cell production in the small intestine was restored to those levels seen in the orally fed rats, moreover, labelling per crypt in the colon was increased to more than twice that of orally fed rats. The location of the mean labelling position and the half maximum labelling position followed the changes in crypt length in the small intestine, but to a lesser extent; thus the growth fraction was significantly increased in the TPN rats in comparison with the orally fed and the URO-EGF treated groups. Similar changes in these positions were seen in the colon, but the growth fraction was much reduced in the URO-EGF treated rats, as a consequence of the large increase in crypt length without a concomitant alteration in label distribution.

Glicentin, an active enteroglucagon, has a significant trophic role on the small intestine but not on the colon in the rat.

BACKGROUND: Many experiments have indicated that the gut glucagons (enteroglucagons) are associated with cell proliferation in the small intestine. However, recent studies have failed to show trophic effects of glicentin (enteroglucagon) on the intestine. AIMS: To examine the effects of glicentin on intestinal proliferation in vivo in the rat. METHODS: Rats were established on total parenteral nutrition for 6 days. Four experimental groups were given daily doses of 1, 4, 20 and 80 microg/rat of glicentin via the jugular vein. Rats fed by total parenteral nutrition and rats fed chow ad libitum were used as controls. Tissues taken from the duodenum, jejunum, ileum and colon were fixed in Carnoy's fluid and microdissected to determine the metaphase arrest scores and crypt fission ratios. RESULTS: The mean metaphase arrest scores per crypt of the small intestine were significantly increased in the rats given 4, 20 and 80 microg of glicentin. These responses were dose-dependent, and were most prominent in the ileum. Crypt fission of the ileum was significantly decreased in the 20 and 80 microg glicentin groups. Glicentin had no effects on proliferation or fission in the colon. CONCLUSIONS: Glicentin is trophic to the rat small intestine, but not the colon.

Systemic effect of peanut agglutinin following intravenous infusion into rats.

BACKGROUND: Ingested peanut agglutinin stimulates colonic proliferation in humans. In rats, ingested peanut agglutinin stimulates hormone release and proliferation in the small and large intestines. Peanut agglutinin is absorbed into the circulation but little is known about the systemic effect of this lectin. Therefore, we studied the effect of intravenous peanut agglutinin on hormone release and intestinal growth. METHOD: Six rats per group received peanut agglutinin infusion at 0, 2, 20 or 200 microg/rat/day for 6 days via the right jugular vein. Organ weights were measured, pancreatic enzymes, DNA, RNA and protein levels were analysed. Plasma hormones were measured by radioimmunoassay. All tissues were examined histologically. Small intestinal and colonic proliferation rates were estimated by metaphase arrest. RESULTS: High-dose peanut agglutinin significantly reduced the wet weight of the stomach by 7% (P < 0.05) and large intestine by 10% (P < 0.05). Peanut agglutinin dose-dependently released enteroglucagon; low-, medium- and high-dose by 64%, 126% (P < 0.01) and 180% (P < 0.01), respectively, and glucagon-like peptide-1 by 127% (P < 0.01), 169% (P < 0.01) and 315% (P < 0.001), respectively. Peanut agglutinin had no effect on cholesystokinin, gastrin or insulin levels. Peanut agglutinin, low-, medium- and high-dose stimulated proliferation in the mid colon by 42% (P < 0.01), 30% and 38%, respectively. Only high-dose peanut agglutinin stimulated proliferation in the distal colon by 54% (P < 0.01). No histological changes were evident in any tissue. CONCLUSION: Intravenous peanut agglutinin released hormones and stimulated colonic proliferation. Proliferation of the small intestine seen after ingestion of peanut agglutinin in previous studies appears to require luminal contact between enterocytes and the lectin. Possible clinical applications include reversal of atrophy during total parenteral nutrition, anastomotic healing after surgery and restoration of mucosa integrity in colitis.

Lectins can reverse the distal intestinal atrophy associated with elemental diets in mice.

BACKGROUND: Elemental diets cause intestinal atrophy and reduced intestinal integrity, which can lead to significant increases in intestinal permeability and bacterial translocation. Recently, several lectins have been shown to have trophic effects on the intestine. AIMS: We examined the effects of concanavalin-A and phytohaemagglutinin on cell proliferation and crypt fission throughout the intestine of mice fed on elemental diets. METHODS: Mice were randomized to chow fed, elemental diet, elemental diet plus concanavalin-A and elemental diet plus phytohaemagglutinin groups. Cell proliferation and crypt fission were estimated in microdissected crypts. Plasma gastrin and enteroglucagon levels were measured by radioimmunoassay. RESULTS: Elemental diet feeding significantly decreased cell proliferation and crypt fission of the middle and distal small intestine and throughout the colon. Phytohaemagglutinin significantly increased the weight of the intestine, but concanavalin-A had little effect. Cell proliferation in the small intestine was significantly increased by both lectins. However, in the stomach and colon, only phytohaemagglutinin increased proliferation. Crypt fission in the colon was dramatically increased by phytohaemagglutinin. Phytohaemagglutinin increased the plasma gastrin level, but not the enteroglucagon level. CONCLUSIONS: Lectins have significant trophic effects on the small intestine and colon of mice fed elemental diets, and these actions vary between different sites in the gastrointestinal tract.

Comparison of the effects of concanavalin-A and epidermal growth factor on epithelial cell proliferation in the rat intestine.

BACKGROUND: Concanavalin-A, the lectin present in Jack beans, binds to mannose- and glucose-containing residues and can interact with the epidermal growth factor receptor and moderate cell proliferation in vitro. AIM: To compare the actions of concanavalin-A and epidermal growth factor on the gastrointestinal tract in vivo. METHODS: Rats maintained on total parenteral nutrition were given intragastric concanavalin-A, intravenous epidermal growth factor or concanavalin-A and epidermal growth factor. Cell proliferation and crypt fission were assayed in 'micro-dissected' crypts. RESULTS: Concanavalin-A and epidermal growth factor both significantly elevated proliferation in the small intestine and colon. No significant interaction between the effects of these two agents was seen, except in the mid small intestine where there was a synergistic interaction. Concanavalin-A had no effect on crypt branching. Epidermal growth factor significantly reduced branching in the distal small intestine and mid colon. CONCLUSION: The effects of the two agents appeared to be separate, except in the mid small intestine where they were additive. This is in marked contrast with the actions reported in vitro, where concanavalin-A is a powerful inhibitor of epidermal growth factor-induced cell proliferation. Concanavalin-A thus has potential for enhancing the functions of the small intestine.

Does the response of the intestinal epithelium to keratinocyte growth factor vary according to the method of administration?

BACKGROUND: Keratinocyte growth factor (KGF) is a potent mitogen and may be of value for the treatment of conditions such as short bowel syndrome and chemotherapy-induced mucositis. However the most efficacious route and method of administration is unclear. METHODS: Rats maintained by total parenteral nutrition (TPN) were given KGF (1 mg/kg/rat/day, i.v.) infused continuously or as a once-daily injection. The same dose was also given s.c. to chow-fed rats. Changes in gut growth were assessed by measurement of wet weight, proliferation (vincristine induced metaphase arrest) and crypt branching index. Changes in gut hormone profile were also determined to examine if any trophic effects were mediated via this mechanism. RESULTS: KGF caused a 70-100% increase in wet weight of the stomach, small and large intestine of TPN-fed rats (P < 0.01) with no significant differences seen between the two methods of administration. The increase in metaphase counts was greatest in the stomach (about seven-fold P < 0.01), but was less pronounced in the distal small intestine and colon (about 50% increase). The trophic effect of KGF was much less prominent in orally-fed rats. Crypt branching index was significantly reduced by KGF in the proximal small intestine of TPN, but not orally-fed rats. Plasma gastrin, PYY, total glucagon, enteroglucagon and GLP-1 all increased by two-three-fold (all P < 0.01) in response to KGF whereas insulin levels fell by about 25% in the TPN group. CONCLUSIONS: The mitogenic action of KGF occurred predominantly in the stomach and proximal small intestine. Its efficacy was less pronounced in orally-fed animals, suggesting KGF may be of greatest benefit in conditions associated with lowered intestinal proliferation. Clinical trials of KGF can probably use single daily i.v. injections without reduction in efficacy.

Soy polysaccharide in an enteral diet: Effects on rat intestinal cell proliferation, morphology and metabolic function.

The present study was conducted to compare the effect of soy polysaccharide (SP)-supplemented and fibre-free enteral diets and a normal chow diet on intestinal and colonic adaptation of rat gastrointestinal morphometrics and cytokinetics. Results showed that the fibre-free diet caused a significant decrease in various gut parameters, such as cell proliferation, tissue wet weight, and intestinal brush border enzymes, when compared to the normal gut of chow-fed rats. However the SP-supplemented enteral diet resulted in a significant improvement in several parameters in most regions along the gastrointestinal tract, when compared to orally-fed animals. These studies demonstrate that significant changes occur in the intestine as a result of enteral diets, with regional variation. Furthermore, the results demonstrate that SP stimulates crypt cell proliferation which could, in part, hasten recovery from intestinal mucosal damage. The addition of SP to enteral diets seems potentially advantageous.

Epidermal growth factor and transforming growth factor-alpha actions on the gut.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are part of a large family of ligands that bind to the epidermal growth factor receptor. While the precise role of these ligands is still unclear, there is little doubt that they play an important role in the development and maintenance of gastrointestinal epithelial continuity. The main ligand for the intestinal EGF receptor (EGFR) may in fact be TGF-alpha, and there is evidence that EGF itself may act as a surveillance factor in adults, only having an effect if the mucosa is damaged.

Conjugated linoleic acids differentially alter polyp number and diameter in the Apc(min/+) mouse model of intestinal cancer.

OBJECTIVE: Dietary conjugated linoleic acids (CLA) have had many health benefits claimed for them, including antineoplastic actions. MATERIALS AND METHODS: The effects of the predominant forms of CLA, namely the c9t11 and t10c12 isomers, or a mixture of these on polyp development, were investigated in the Apc(Min/+) mouse. CLAs have also been linked to altered rates of cell renewal and cell proliferation so this was also studied, as was a further means of increasing tissue mass, namely crypt fission. RESULTS: The stomach and small intestine were significantly heavier in the t10c12, and in the mixture-treated groups (P < 0.001). Crypt fission was increased in the middle small intestine by the t10c12 diet while colonic weight was reduced by c9t11 provision and crypts were 20% shorter. The t10c12 and the mixture significantly reduced polyp number in the proximal small intestine but they increased polyp diameter in the middle and distal small intestine, to an extent that the polyp burden was significantly increased at these sites. All CLAs significantly reduced polyp number in the colon, but the mixture significantly increased polyp diameter in the colon. CONCLUSION: Increased polyp diameter associated with t10c12 diet and especially with the mixture is a cause of concern, as this is the commercially available form. The naturally occurring isomer, c9t11 decreased colonic polyp number and did not increase diameter, suggesting that this natural isomer is the most likely to be protective.

The development of duodenal microadenomas in FAP patients: the human correlate of the Min mouse.

UNLABELLED: The morphological changes associated with the adenoma-carcinoma sequence are well documented in the colorectum. Small intestinal carcinogenesis is thought to progress through a similar adenoma-to-carcinoma pathway, but there is a relative dearth of studies examining the associated morphological changes. The best-known mouse model of intestinal neoplasia, the multiple intestinal neoplasia (Min) mouse, has been criticized as a genetic model of intestinal neoplasia, as the majority of its tumours occur in the small intestine. We examined pancreatico-duodenal resection specimens from seven familial adenomatous polyposis (FAP) patients. Serial sections of these were stained with haematoxylin and eosin for beta-catenin and its downstream target CD44, for BMPR1a, lysozyme, carbonic anhydrase II, and with MIB-1. Individual dysplastic crypts were isolated and mutations in the FAP (APC) gene compared between the top and bottom of the crypt. We found that: (a) duodenal microadenomas are extremely common in FAP patients; (b) these grow in the core of duodenal villi, forming lesions similar to those described in the Min mouse; (c) many lesions arise as monocryptal adenomas and grow by a process of crypt fission and branching; (d) migrating adenomatous cells lose their dysplastic phenotype as they migrate up the crypt villous axis; and (e) Paneth cells lose positional information. IN CONCLUSION: (a) the morphological similarity of adenomas in the Min mouse and human suggest the Min mouse is a good model of FAP; (b) duodenal adenomas in FAP originate in monocryptal adenomas and follow the 'bottom-up' rather than the 'top-down' model of morphogenesis; (c) early microadenomas show evidence of cellular differentiation; (d) defects in the positioning of Paneth cells suggests disruption of the EphB2:EphB3 receptor system.

Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer.

The Apc(Min/+) mouse model is a clinically relevant model of early intestinal cancer. We used AZD2171, an oral, highly potent and selective vascular endothelial growth factor (VEGF) signaling inhibitor, to investigate the role of VEGF receptor-2 (VEGFR-2) signaling in adenoma development and growth in Apc(Min/+) mice. AZD2171 (5 mg/kg body wt/day) was administered once daily for 28 days to 6-week-old (early-intervention) or 10-week-old (late intervention) mice. In the early-intervention study, AZD2171 reduced the number of macroscopic polyps in the small bowel and colon. Macropolyp diameter was lower in the small bowel, but remained unchanged in the colon. In animals receiving AZD2171, microscopic evaluation of the small intestine showed a significant reduction in the number of larger lesions. In the late-intervention study, AZD2171 treatment reduced macropolyp diameter (but not number) in the small intestine. Microscopic analysis revealed that AZD2171 significantly reduced the number of larger micropolyps in the small bowel, with no large micropolyps present in the colon. AZD2171 treatment had no effect on microvessel density or localization of beta-catenin staining in adenomas or non-tumor intestinal tissue, but significantly reduced the number of cells expressing VEGFR-2 mRNA. In conclusion, the effects of AZD2171 in the small intestine of Apc(Min/+) mice are consistent with an antiangiogenic mechanism of action, limiting growth of adenomas to < or =1 mm. These data also suggest that an early step in adenoma development may depend on VEGFR-2 signaling. Together, these results indicate that VEGFR-2 signaling may play key roles in the development and progression of intestinal adenomas.

Regulation of stromal cell cyclooxygenase-2 in the ApcMin/+ mouse model of intestinal tumorigenesis.

Cyclooxygenase-2 (Cox-2) is expressed predominantly by stromal cells in intestinal adenomas from the Apc(Min/+) mouse model of familial adenomatous polyposis. We investigated the mechanistic basis of stromal cell Cox-2 expression in Apc(Min/+) mouse adenomas, as well as Cox-2 expression and activity in histologically normal (HN) Apc(Min/+) mouse intestine, in order to gain further insights into regulation of Cox-2 as a potential chemoprevention target. Upregulation of Cox-2 in intestinal tumours is not an intrinsic feature of Apc(Min/+) macrophages as bone marrow-derived Apc(Min/+) macrophages did not exhibit an abnormality in Cox-2 expression or activity. Intestinal permeability to lactulose or mannitol was similar in Apc(Min/+) mice and wild-type littermates, implying that macrophage activation by luminal antigen is unlikely to explain stromal cell Cox-2 induction. Moreover, stromal cells exhibited differential expression of Cox-2 and inducible nitric oxide synthase, suggesting 'alternative' (M2) rather than 'classical' (M1) macrophage activation. Flow cytometric sorting of isolated stromal mononuclear cells (SMNCs), on the basis of M-lysozyme and specific macrophage marker expression, demonstrated that macrophages, neutrophils and non-myelomonocytic cells all contributed to lamina propria prostaglandin (PG) E(2) synthesis. However, the majority of PGE(2) synthesis by macrophages was via a Cox-2-dependent pathway compared with predominant Cox-1-derived PGE(2) production by non-myelomonocytic cells. SMNCs from HN Apc(Min/+) intestinal mucosa exhibited similar levels of Cox-2 mRNA and protein, but produced more Cox-2-derived PGE(2) than wild-type cells at 70 days of age. There was an age-dependent decline in PGE(2) synthesis by Apc(Min/+) SMNCs, despite tumour progression. These data suggest that other Cox-2-independent factors also control PGE(2) levels during Apc(Min/+) mouse intestinal tumorigenesis. Regulation of macrophage Cox-2 expression and other steps in PGE(2) synthesis (e.g. PGE synthase) are valid targets for novel chemoprevention strategies that could minimize or avoid systemic COX-2 inhibition.