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

Abstract. 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.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell kinetics were investigated in the descending colon of the rat. The number of cells per crypt was found to be approximately 625, with 33 cells per cell column and 19 cell columns per crypt circumference. The growth fraction of the colonic crypt was 0.42, and proliferating cells were situated largely in the lower half of the crypt. The cell cycle time was 50.5 h, with values for the G1, S and G2 phases of 40.0, 7.6 and 2.9 h respectively. Cell migration studies showed that it took 60-72 h for a cell to migrate from the upper border of the proliferative cell compartment in the crypt to the luminal surface of the colon. Data were also obtained from continuous labelling with tritiated thymidine and from studying the circadian rhythm of proliferative activity, which suggest that the cells in the bottom of the crypt may constitute a separate, more slowly cycling (stem)cell compartment.     

A cytokinetic study of small-intestinal and colonic mucosa after resection of 70% of the small intestine

Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied. In the distal ileum there is a significant reduction in cell cycle time (Tc) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in Tc at these sites, but there does appear to be a significant reduction in Tc on the part of the cells in the stem-cell zone at the crypt base. In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

A Cytokinetic Study of Small-Intestinal and Colonic Mucosa After Resection of 70% of the Small Intestine

Abstract. Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied.
In the distal ileum there is a significant reduction in cell cycle time (T_c) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in T_c at these sites, but there does appear to be a significant reduction in T_c on the part of the cells in the stem-cell zone at the crypt base.
In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

THE EFFECT OF TRANSPOSITION TO JEJUNUM ON EPITHELIAL CELL KINETICS IN AN ILEAL SEGMENT

Epithelial cell kinetics were studied in an ileal segment after transposition to proximal jejunum. The number of cells per villus column in the transposed ileum increased after 4-7 days to reach values normal for jejunum after 14-30 days. This increase was accompanied by a simultaneous increase in the number of cells per crypt column up to 130% of values in jejunum and ileum in situ. The percentage of labelled crypt cells, after labelling with ³H-thymidine, and the relative size of the proliferative cell compartment in the crypt in the transposed ileum did not differ from values in the ileum in situ at any time interval after surgery. The total proliferative activity per crypt, which was determined by scintillation counting of isolated crypts after ³H-thymidine labelling, increased two-fold from 7 days after surgery. Cell migration studies showed that the increase in the number of villus cells was probably not caused by a change in the life span of the epithelial cells. It seems that the increase in the number of villus cells in ileal epithelium after transposition to proximal jejunum is brought about by an enlargement of the crypt, while the relative size of the proliferative cell compartment in the crypt remains unchanged.     

Epithelial cell kinetics in the descending colon of the rat

The influence of experimental bypass on the epithelial cell kinetics in the rat descending colon was studied. It was found that the number of cells per crypt was markedly reduced at 6 weeks after bypass. The percentage of labelled crypt cells, 1 h after 3HTdR, and the distribution of labelled cells in the crypt was normal. Also the life span of the epithelial cells was the same in control and bypassed colon. The response of crypt cell proliferation to ischaemia-induced cell loss in the bypassed descending colon was similar to the one previously described for normal descending colon. This indicates that the absence of the normal luminal contents does not result in a different response of colonic crypts to induced cell loss. Furthermore, it was found that the number of cells per crypt and the proliferative activity did not change in the transverse colon after temporary ischaemia of the bypassed descending colon. This indicates that the increase in crypt cell proliferation after ischaemia-induced cell loss is a local response.     

PCT及CRP在急性肠梗阻大鼠血清中的表达水平及其临床意义

目的:探讨降钙素原(Procalcitonin,PCT)及C反应蛋白(C-reactive protein,CRP)在急性肠梗阻大鼠血清中的水平及其临床意义。方法:将83只Wistar大鼠分为对照组(n=13)、假手术组(n=35)和急性肠梗阻组(n=35)。对照组大鼠采集标本后处死,肠梗阻组行开腹手术结扎回肠末端,假手术组仅行开腹手术。检测8 h、24 h、48 h、72 h及96 h血清PCT及CRP水平,观察急性肠梗阻大鼠回肠组织的病理学改变情况。结果:假手术组PCT与CRP水平在术后24 h内显著升高,48 h至96 h逐渐下降;各时间点PCT水平明显高于对照组,而CRP水平在实验结束时已恢复至正常水平。肠梗阻组PCT和CRP水平在各时间点均明显高于对照组,并逐渐增加,到实验结束时达到高峰;肠梗阻组PCT和CRP水平在48 h-96 h均显著高于假手术组,差异具有统计学意义(P0.05)。组织病理学检查显示,对照组大鼠肠壁粘膜结构正常,假手术组可见轻度病理改变,肠梗阻组大鼠回肠组织可见粘膜结构明显破坏,绒毛坏死,严重水肿和炎症细胞浸润。结论:血浆PCT和CRP水平能够反映肠梗阻的状态和肠粘膜受损程度。     

The effect of single and of multiple doses of prednisolone tertiary butyl acetate on cell population kinetics in the small bowel mucosa of the rat.

The effect of single and of multiple doses of prednisolone upon cell population kinetics in the rat jejunal crypt was investigated, using autoradiography and stathmokinetic techniques with vincristine. Single injections of prednisolone (2.5 mg/kg body weight) induced a depression both flash thymidine labelling and mitotic indices; this change was shown to be due to a decreased cell production rate. Recovery of these proliferative indices occurred over seven days after injection; measurement of crypt size parameters showed a transient decrease in crypt population. Multiple daily injections of prednisolone (1 mg/kg body weight) produced a more sustained decrease in labelling and mitotic indices, which lasted as long as injections were continued (7 days); stathmokinetic techniques showed decreases in cell production rates, and the crypt population was also depressed throughout this period. It is concluded that prednisolone depresses cell proliferative rates in rat jejunal mucosa.     

Taenia taeniaeformis: colonic hyperplasia in heavily infected rats

Only one study previously mentioned the involvement of colon during Taenia taeniaeformis larvae infection in rats with inconsistent occurrence of lesions. Present study aimed to determine the consistency of histopathologic changes in colonic epithelia, and the proliferation of mucosal cells through BrdU and PCNA immunohistochemistry. Results demonstrated that crypt hyperplasia of the colon was found in all infected rats, although variable in degree even in a single tissue section. Cystic cavities were frequently seen in severely hyperplastic mucosa. Proliferative zone lengths were significantly increased and PCNA positive cells were observed throughout the colonic crypt lengths at 9 but not at 6 weeks post infection. Cell proliferation involving the major types of cells in the epithelial colon was also increased in infected rats at 9 weeks post infection, with labeling indices significantly greater than the control rats throughout the BrdU time course labeling. Findings suggested that massive increases in epithelial cells and depth of colonic crypts were due to a remarkable increase in cell proliferation. The study concluded that enteropathy in the colon during T. taeniaeformis infection could be consistently observed in heavily infected rats.     

The effect of bile and of sodium taurocholate on the epithelial cell dynamics of the rat small intestine.

The absence of bile from the intestinal lumen of rats for a period of 48 hr led to: a decreased proliferative cell pool, reduced cell shedding, and a 50% decrease of the labeling index in the ileum. The constant duodenal perfusion of Na taurocholate for periods of 48, 60, 72, and 96 hr in animals with a biliary diversion was associated with: deepening of crypts and decreased crypt/villus ratios as well as with acceleration of the epithelial cell migration rate on the crypt-villus units. The data suggest that bile and bile acids constitute important regulatory factors influencing enterocyte proliferation, migration and loss.     

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.     

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat. A study of enzymes bound to subcellular organelles

The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus.The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R).Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β-N-acetylglucosaminidase) are not affected by changing crypt cell kinetics.Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.     

Quantitative contribution of factors regulating rat colonic crypt epithelium: role of parenteral and enteral feeding, caloric intake, dietary cellulose level and the colon carcinogen DMH

To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2-dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9.5 mg kg-1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.     

Epithelial cell proliferation and uptake of radiolabeled urogastrone in the intestinal tissues following abdominal irradiation in the mouse

We have evaluated the rate of crypt cell production and uptake of radiolabeled recombinant human urogastrone (125I-rhUG) in the intestinal tissues of the mouse at 3, 5, 7, 9, and 12 days following irradiation of the abdomen with 9 Gy. At autopsy, the animals were injected intraperitoneally with 1 microgram/g body weight of the metaphase arrest agent, vincristine sulfate, and 25 muCi of 125I-rhUG (specific activity 1.7 muCi/micrograms) to quantify the rate of crypt cell production and uptake of radiolabeled urogastrone, respectively. The results indicated that the rate of crypt cell production was increased significantly in the irradiated animals compared to the unirradiated animals and showed a peak on the 3rd and 5th postirradiation days in small intestine and colon, respectively. The uptake of 125I-rhUG was increased significantly on the 3rd postirradiation day in the intestinal tissues but showed a bimodal pattern with peaks on the 3rd and 9th postirradiation days. These results suggest that there may be a close association between epithelial cell proliferation and uptake of 125I-rhUG, particularly in the early part of recovery of intestinal mucosa following irradiation. However, these data do not discriminate whether the increased uptake of 125I-rhUG is the cause or the effect of proliferation induced by an irradiation stimulus. Further analysis also revealed that there was no relationship between crypt depth and 125I-rhUG uptake. However, crypt depth was inversely correlated with villus height in the proximal small intestine but not in the ileum. Villus height was correlated inversely with 125I-rhUG uptake in the ileum and jejunum but not the duodenum. The rate of crypt cell production was strongly correlated with crypt depth throughout the intestine and inversely correlated with villus height. This suggests that villus-to-crypt inhibitory feedback may be a primary regulator of cellular proliferation in the crypts and the association of 125I-rhUG uptake with proliferation indirectly reflects this interaction.     

A possible mechanism of induction and translocation into blood stream of rat alkaline phosphatase activity by bile duct ligation

We investigated the effects of bile duct ligation on alkaline phosphatase (ALP) activities in liver, calvarium, duodenum, and ileum in rats and its possible mechanism of action. ALP isozyme activities in the ligated rats were significantly elevated in the liver and duodenum, while those in the ileum and calvarium were markedly decreased. The ALP isozyme activity elevated by the ligation was obviously suppressed by prior administration of indomethacin, an inhibitor of prostaglandin synthesis. Moreover, phorbol ester also elevated the ALP activity as well as the phosphatase level in the ligated rat. However, other drugs, such as an inhibitor of protein kinase C and calmodulin, showed different effects: calmodulin stimulated an 11.0-, 1.3-, or 1.5-fold increase in ALP activity in the ileum, duodenum, or calvarium, respectively; whereas the hepatic enzyme activity was not affected. The induction by calmodulin was markedly different from that by the ligation. Moreover, imipramine, an inhibitor of protein kinase C, had little effect. These results suggest that prostaglandin is a possible ALP inducer in ligated rats, probably working by elevating the cAMP level. On the other hand, the ligation induced simultaneously de novo synthesis of the membranous and soluble ALP isozymes; and the release rate of the soluble enzyme was greater than that of the membranous isozymes, indicating that the soluble enzyme might be a main source of the induced serum ALP. Lectin affinity chromatography indicated that the soluble enzyme or induced serum enzyme may contain more fucose than that of the membranous one, suggesting that the sugar moiety in the ALP molecule may relate to the clearance of ALP from or its release into the circulation.     

The effects of starvation and refeeding on intestinal cell proliferation in the mouse

The effects of starvation and refeeding on intestinal cell proliferation were studied in four sites of the mouse intestine. Control mice were studied at different times of day in order to compensate for any circadian variations in proliferation. A circadian rhythm in crypt cell production rate was observed in all the sites of the small intestine and colon, and this rhythm appeared to be entrained to the food intake. The fractional crypt cell production rate decreased in all sites of the intestine after 24 h starvation, and remained low until 9 h after refeeding, when there was a marked increase in the crypt cell production rate of all the small intestinal sites, especially the proximal sites. There was little change in colonic crypt cell production rate until 12 h after refeeding, when there was a large increase in cell production. The crypt cell production rate of all sites then returned to control values for the remainder of the investigation. Crypt cell number decreased after refeeding and villus cell number increased, however a similar effect was observed in the control animals, nevertheless the changes in villus cell population of the refed mice occurred before any increase in crypt cell production, suggesting that cell migration from crypt to villi is not immediately dependent on cell proliferation.     

Influence of triamcinolone and somatostatin on morphometric parameters of cultured intestinal mucosa

Summary Mucosal biopsies from rabbit ileum were organ cultured for 24 h. The influence of triamcinolone and somatostatin on vilus height and diameter as well as crypt depth and the number of mitoses was measured at various times during 24 h of culture as indices of cell proliferation and tissue maintenance. It could be shown that triamcinolone reduced cell proliferation slightly but preserved mucosal structure in organ culture. Somatostatin inhibited crypt cell proliferation, without any effect on other morphological parameters.     

Aspirin suppresses 1,2-dimethylhydrazine-induced alteration of proliferative parameters in rat colonic crypts

Abstract. Human epidemiological reports and rodent experimental research data indicate a possible chemopreventive effect of regular aspirin use for decreasing risk of colon and rectum cancer incidence and mortality. We have previously demonstrated that aspirin can significantly suppress proliferative parameters in normal rat colonic epithelium when examined 24 h following an acute or chronic course of aspirin administration. To investigate whether aspirin would effectively suppress known carcinogen-induced changes in colonic epithelium, rats were given single s.c. injections of either aspirin (50 mg/kg bw) or saline on days 1–3 and either 1,2-dimethylhydrazine (DMH; 12 mg base/kg bw) or DMH vehicle on day 4 of each week for eight consecutive weeks. Rats were sacrificed 4 days after the last aspirin dose and 3 days after the last DMH or DMH vehicle dose. Using the proliferative biomarkers of proliferating cell nuclear antigen positive cells per midaxial crypt section (SCC), crypt proliferative zone height (PZ), crypt differentiated zone height (DZ), and total crypt height (CH), it was found that aspirin does suppress DMH-induced increases in SCC, PZ and CH. The findings demonstrate that aspirin has a long term (i.e. several days) protective effect against early carcinogen-induced proliferative changes in rat colonic crypts which may help account for aspirin's chemopreventive action against colon cancer.     

Acute and subacute effects of thiamine deficiency on the morphometry and cell kinetics of jejunal and ileal epithelial cells

The effects of acute and subacute thiamine deficiency on jejunal and ileal epithelial cells were studied in rats, using crypt and villus cell population, crypt cell production per crypt (CCPC), crypt growth fraction (Ip) and crypt cell cycle time (Tc) as parameters. In acute thiamine deficiency there was marked jejunal hypoplasia of the crypt and villus, but in the ileum there was hypoplasia only of the crypt. The jejunal epithelium of the subacute thiamine deficiency (STD) group showed no morphometric changes. In contrast, in the ileal epithelium of STD rats there was decreased crypt depth and villus cell population. Thiamine deficiency had no significant effect on CCPC, Ip and Tc.