Alterations In Gastrointestinal Steady-State Kinetics Associated With the Growth of Experimental Tumours

Changes in the growth kinetics of the intestinal epithelium were observed in mice bearing the Lewis lung carcinoma and the T1699 mammary adenocarcinoma and in rats bearing the H-4-II-E₂ hepatoma. Proliferative activity in the jejunal tissue was markedly depressed with increasing tumour burden. Simultaneously, a significant reduction in total crypt cellularity occurred, followed by a reduction in villus height. While the total number of proliferative cells per crypt decreased, the relative proliferative compartment within the shrinking crypt increased. the rate of mucosal DNA synthesis remained constant during the initial cytokinetic changes, falling only after proliferative activity of the intestine was reduced to less than 50% of control levels. No general correlation could be drawn from the three tumour models studied between the level of gastrointestinal proliferation and tumour size, tumour growth rate or loss of weight by the tumour-bearing animals. However, intestinal proliferation was reduced by 50% when the tumour burden for each of the three tumours reached 6–8% of the host animal weight.     

A study of diurnal proliferative activity in tumour and small intestine of C3H mice bearing a transplanted mammary carcinoma

Diurnal changes in proliferative activity were investigated in tumour and small intestinal epithelium of mice bearing a transplanted mammary carcinoma. In addition to mitotic and labelling index studies, the metaphase-arrest technique with vincristine (VCR) was employed. In the tumour there was no clear evidence of a significant diurnal rhythm in proliferative activity but in the small intestinal epithelium such a rhythm was clearly demonstrated. A higher cell production rate (kB) measured by metaphase-arrest and higher labelling and mitotic indices were seen in the mid to late part of the dark period. The peak mitotic index was seen 3 to 6 h after the labelling peak in the small intestine. The basal third of the crypt which is believed to include the stem cell compartment of this tissue showed larger diurnal fluctuations in both labelling index and kB than the rest of the proliferative compartment.     

Simulation modelling of the postradiation restoration of the crypt-villus system

Basic principles have been developed for a discrete stochastic simulation model of an elementary proliferative unit of the intestinal epithelium, a "crypt-villus" system. The analysis of the results obtained after a single exposure of the animal's abdomen to 3 and 6 Gy radiation has demonstrated that the dynamics of the number of cells that synthesize DNA in a small intestine crypt of exposed mice depends on the rate of radiation damage repair (50 to 100 h following irradiation). The rate of repair after 6 Gy irradiation is 1.5 times lower that after 3 Gy. The changes in the shape of the labeled mitoses curve, followed up during the postirradiation recovery of the intestinal epithelium, may occur with the time parameters of the cell mitotic cycle being invariable.     

The influence of 400 r x-irradiation on the number and the localization of mature and immature goblet cells and Paneth cells in intestinal crypt and villus.

The influence of 400 R X-irradiation on the localization and the number of mature and immature goblet cells and Paneth cells in rat duodenal epithelium has been studied. At short times after irradiation, when the total proliferative activity in the crypts of Lieberkuhn is reduced, the proportion of mature and immature goblet cells of the total number of crypt cells was increased; also an absolute increase in the number of goblet cells in the crypts was found. The immature goblet cells were localized in the lower half of the crypt as in control animals, whereas the number of the mature cells increased over the whole crypt length. When the proliferative activity of the crypt cells increases again from 12 to 48 hr after irradiation the number of both types of goblet cells decreases. Between 48 and 72 hr, when the whole crypt is involved in proliferation, a second increase of both types of goblet cells was found. However, the localization of the immature goblet cells is no longer restricted to the lower half of the crypt but they also appear at the higher cell positions. On the villus no immature goblet cells were found and the changes in the numbers of mature goblet cells do reflect the changes induced by irradiation in the goblet cell population in the crypt. The absolute number and localization of Paneth cells did not change under the experimental conditions. The findings are discussed in relation to cell proliferation and differentiation processes in intestinal crypts.     

CHANGES IN GROWTH KINETICS OF JEJUNAL EPITHELIUM IN MICE MAINTAINED ON AN ELEMENTAL DIET

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurrred 16 hr later in diet-fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet-fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.     

Changes in growth kinetics of jejunal epithelium in mice maintained on an elemental diet.

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurred 16 hr later in diet-fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet-fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.     

THE INFLUENCE OF 400 R X-IRRADIATION ON THE NUMBER and THE OCALIZATION OF MATURE and IMMATURE GOBLET CELLS and PANETH CELLS IN INTESTINAL CRYPT and VILLUS

The influence of 400 R X-irradiation on the localization and the number of mature and immature goblet cells and Paneth cells in rat duodenal epithelium has been studied. At short times after irradiation, when the total proliferative activity in the crypts of Lieberkiihn is reduced, the proportion of mature and immature goblet cells of the total number of crypt cells was increased; also an absolute increase in the number of goblet cells in the crypts was found. The immature goblet cells were localized in the lower half of the crypt as in control animals, whereas the number of the mature cells increased over the whole crypt length. When the proliferative activity of the crypt cells increases again from 12 to 48 hr after irradiation the number of both types of goblet cells decreases. Between 48 and 72 hr, when the whole crypt is involved in proliferation, a second increase of both types of goblet cells was found. However, the localization of the immature goblet cells is no longer restricted to the lower half of the crypt but they also appear at the higher cell positions. On the villus no immature goblet cells were found and the changes in the numbers of mature goblet cells do reflect the changes induced by irradiation in the goblet cell population in the crypt. The absolute number and localization of Paneth cells did not change under the experimental conditions. The findings are discussed in relation to cell proliferation and differentiation processes in intestinal crypts.     

VARIATION IN THE DURATION OF MITOSIS IN THE CRYPTS OF LIEBERKUHN OF THE RAT; A CYTOKINETIC STUDY USING VINCRISTINE

The variation in the duration of mitosis ( t _m) with cell position in the small intestinal crypts of the adult rat has been measured by a stathmokinetic technique using vincristine. The value for the whole crypt column was 0.43 hr, or 26 min. At the bottom of the crypt t _m was in excess of 1 hr, but rapidly decreased and throughout the greater part of the proliferative compartment was between 0.40 and 0.50 hr. At the top of the proliferative compartment an increase in t _m was demonstrated.
If the value of 0.43 hr for the whole crypt column is correct, then one argument for postulating the formation of metabolic DNA during differentiation in the small bowel epithelium of the rat becomes invalid. Variations in t _m within the crypt have been shown to increase the values of cell velocity obtained from cumulative birth rate diagrams. Finally further evidence has been presented for the existence of a slowly dividing subpopulation of cells at the base of the crypt. These cells may be important in crypt repopulation after damage with phase specific anti-tumour drugs.     

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

THE EFFECT OF CONTINUOUS IRRADIATION ON CELL PROLIFERATION AND MATURATION IN SMALL INTESTINAL EPITHELIUM

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with ³H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36–48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

Mathematical modeling of the dynamics of the intestinal epithelium in non-irradiated and irradiated mammals]

A mathematical model has been developed to describe the dynamics of the crypt-villus system of the small intestine epithelium in nonirradiated and chronically irradiated mammals. The model involves the chalone mechanism of regulation of crypt cell reproduction and represents a system of nonlinear differentiation equations. The model presents the dynamics of the small intestine epithelium in nonirradiated animals, including stable fluctuations of the concentrations of crypt and villus cells (the limited cycle), and simulates quantitatively the impairment of the intestinal epithelium in small laboratory animals subjected to long-term irradiation.     

Late effects in the mouse small intestine after a clinically relevant multifractionated radiation treatment

Late radiation effects were investigated in the mouse small intestine after a daily fractionated radiation treatment. Mice were given 14 X 3 Gy in 2 weeks over a partial abdominal irradiation field. There was evidence for late injury in the intestinal epithelium, the submucosa, and the subserosa. Late damage in the epithelium was shown histologically by a reduced crypt number and villus atrophy at 3 and 6 months but not at 24 h after the end of treatment. The reduction in crypt number was significant in the ileum at 3 and 6 months after irradiation: 100 +/- 4 and 98 +/- 5 (SEM) per circumference, respectively, versus 132 +/- 3 and 146 +/- 6 in age-matched controls (P less than 0.01, t test). The mitotic activity in the crypts of the irradiated animals was significantly increased at all investigated times, suggesting a prolonged but insufficient compensatory response to maintain the mucosal integrity. The repercussion on intestinal epithelial function was, at least in part, reflected by a progressively reduced body weight gain up to 5 g at 3 months after treatment. The ability of the surviving crypt stem cells to form microcolonies after irradiation, however, was not impaired. Evidence for injury in the submucosa was provided from macroscopic and histological examination. Macroscopically, at 6 months after treatment, narrowed and rigid bowel segments surrounded by fibrotic adhesions were observed, causing partial intestinal obstruction. In addition, sometimes focal areas of hemorrhage and infarction in small bowel segments were present. Histologically, diffuse and pronounced submucosal edema without increased fibrosis was seen, together with markedly dilated small blood vessels in focal areas of macroscopic intestinal infarction. The intestinal perfusion, as assessed by 86Rb extraction, was significantly but transiently reduced at 3 months after irradiation. These data suggest mainly late effects in the small intestine after this daily fractionated irradiation treatment. The reduced number of epithelial cells and the submucosal edema are possibly mediated by radiation injury in the intestinal microvasculature.     

Effect of syngeneic thymocytes on the proliferation of the small intestine epithelium in mice

The effect of syngeneic thymocytes on the proliferative compartment of the small intestinal epithelium was studied in CBA mice. In mice that intravenously received thymus cells, the proliferative enterocyte activity was depressed, the proliferative zone reduced and the maturation zone enlarged. The data obtained indicate that medullary thymocytes are responsible for this effect. The regulatory role of the immune system with regard to cell proliferation is discussed.     

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.     

The effect of T-lymphocytes on epithelial proliferation of the jejunal mucosa in thymectomized mice

Following 1.5 month period after thymectomy of adult mice the crypt 3H-thymidine labelling index is decreased and the proliferative zone is reduced. Analysis of labeled cell distributions in the crypt revealed that thymectomy resulted in the initiation of enterocyte entering a maturation phase in the site shifted to the crypt base. Syngeneic T-lymphocytes injected intravenously to thymectomized mice increased the labeling index, but exerted no effect on the size of proliferative zone. The proliferative changes described were not due to changes in the number of divisions of rapidly proliferating enterocytes. The role of T-lymphocytes in modulation of intestinal epithelium proliferation and steady state maintenance is discussed.     

Reduction of spontaneous and irradiation-induced apoptosis in small intestine of IGF-I transgenic mice

Insulin-like growth factor I (IGF-I) may promote survival of putative stem cells in the small intestinal epithelium. Mitosis and apoptosis were quantified in crypts of nonirradiated and irradiated IGF-I transgenic (TG) and wild-type (WT) littermates. The mean apoptotic index was significantly greater in WT vs. TG littermates. After irradiation, apoptotic indexes increased, and WT mice showed a more dramatic increase in apoptosis than TG mice at the location of putative stem cells. After irradiation, no mitotic figures were observed in WT crypts, whereas mitosis was maintained within the jejunal epithelium of TG mice. The abundance and localization of Bax mRNA did not differ between nonirradiated littermates. However, there was more Bax mRNA in TG vs. WT mice after irradiation. Bax mRNA was located along the entire length of the irradiated crypt epithelium, but there was less Bax protein observed in the bottom third of TG mouse crypts compared with WT littermates. IGF-I regulates cell number by stimulating crypt cell proliferation and decreasing apoptosis preferentially within the stem cell compartment.     

CONTROL OF EPITHELIAL CELL PROLIFERATION IN THE SMALL INTESTINAL CRYPT

A heat labile factor which has been shown to inhibit proliferative activity in crypt epithelium both in rat jejunum in vivo and in explants of rat jejunum maintained in organ culture has been prepared from the soluble fraction of homogenized epithelial cells isolated from rat small intestinal crypts. The factor appears to have tissue specificity, for it has no influence on epithelial cell proliferation in colonic crypts, oesophagus or skin. Extracts of rat intestinal villous cells prepared using identical techniques were without effect on proliferative activity of small intestinal crypt epithelium.
Isoprenalin, which was also found to suppress cell proliferation, did not potentiate the effect of the factor and its effects were evanescent.     

Cellular apoptosis]

The interrelations between lymphocytes and intestinal epithelium cells after total X-ray irradiation (15 Gy) were investigated autoradiographically in CBA mice. The labelled lymphocytes injected in the irradiated animals migrated in intestinal epithelium, the label being present within vacuoles of the crypt cells and later in their nuclei. The trophic function of lymphocytes and the nature of the so-called apoptosis are discussed.     

Teduglutide ([Gly2]GLP-2) protects small intestinal stem cells from radiation damage

Glucagon-like peptide-2 and its dipeptidyl peptidase (DP-IV) resistant analogue teduglutide are trophic for the gastrointestinal epithelium. Exposure increases villus height and crypt size and results in increased overall intestinal weight. As these effects may be mediated through stimulation of the stem cell compartment, they may promote intestinal healing and act as potential anti-mucositis agents in patients undergoing cancer chemotherapy. A study was initiated to investigate the protective effects of teduglutide on the murine small intestinal epithelium following gamma-irradiation using the crypt microcolony assay as a measure of stem cell survival and functional competence. Teduglutide demonstrated intestinotrophic effects in both CD1 and BDF1 mouse strains. In BDF1 mice, subcutaneous injection of GLP-2 or teduglutide (0.2 mg/kg/day, b.i.d.) for 14 days increased intestinal weight by 28% and resulted in comparable increases in crypt size, villus height and area. Teduglutide given daily for 6 or 14 days prior to whole body, gamma-irradiation significantly increased crypt stem cell survival when compared with vehicle-treated controls. The mean levels of protection over a range of doses provided protection factors from 1.3 to 1.5. A protective effect was only observed when teduglutide was given before irradiation. These results suggest that teduglutide has the ability to modulate clonogenic stem cell survival in the small intestine and this may have a useful clinical application in the prevention of cancer therapy-induced mucositis.     

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.