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.     

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 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.     

PROLIFERATION KINETICS IN EPITHELIUM OF GUINEA-PIG COLON III. DISTRIBUTION OF BLOOD CAPILLARIES ALONG THE CRYPT

The colonic blood vessels of the guinea-pig were infused with carmine-gelatine and the occurrence along the crypt of mucosal capillaries containing the dye was examined. The pattern of capillary distribution along the crypt is inhomogeneous but is similar for short, medium and long crypts. Capillaries occur least frequently at the bottoms of crypts, that is within the lowest 0.05% of the crypt length. The frequency rises significantly between 0.05 and 0.40 of the crypt length; this rise is accompanied by an increase in the number of DNA-synthesizing cells. A significant fall in capillary frequency along the crypt between 0.35 and 0.45 of crypt length coincides with the beginning of a decrease in the number of DNA-synthesizing cells. The highest frequency of capillaries occurs in the upper 0.20 of the crypt length and beneath the lining epithelium.     

CHANGES IN INTESTINAL CELL KINETICS IN THE SMALL INTESTINE OF LACTATING MICE

The enlargement of the small intestine of mice during lactation is due, at least in part, to hyperplasia in the mucosal crypts and villi. The number of cells per crypt increases by 130% and the cell production rate by 63% after 15 days of lactation. These parameters were measured from crypt squashes and sections using both double-label and PLM techniques. Neither the numbers of crypts and villi in the small intestine nor the turnover time of post-mitotic cells on the villi changed. A number of factors appear to act in concert during lactation to trigger this increase in epithelial cell number in the small intestine. The experiments reported suggest a role for the increased quantity of food consumed by the lactating animal, for changing hormonal levels, and for the increased demands placed on the body by milk production.     

MODE OF GROWTH OF THE JEJUNAL CRYPT CELLS OF THE RAT: AN AUTORADIOGRAPHIC STUDY USING DOUBLE LABELLING WITH 3H- AND 14C-THYMIDINE IN LOWER AND UPPER PARTS OF CRYPTS

The frequency distribution of cells through the mitotic cycle in lower and upper portions of jejunal crypts of the rat was examined by the ³H-¹⁴C-thymidine double labelling technique. Isolated crypts were cut perpendicular to the longitudinal axis so that the percentage of cells in the lower portion varied from 16 to 74 %. The lower and upper portion of the same crypt were squashed separately on one microscope slide and the number of ³H- and ¹⁴C-only labelled cells were scored to determine the flow rate into and out of S for the two portions. The mitotic cycle and its phases of the crypt epithelial cells were also determined. For lower portions of crypts which contained less than 40 % of the total cell number in that crypt the flow rate into S was about 1–7 times that of the flow rate out of S indicating that nearly every mitosis in this region produced two proliferative daughter cells. As the proportion of cells in the lower part of the crypt increased the quotient of the flow rate into S divided by the flow rate out of S decreased, and approached the steady state value of 1 0 in lower portions containing 60–74 % of the cells. For upper portions of crypts which contained less than 40% of the total crypt cells the flow rate into S was about 0 2 times that of the flow rate out of S, indicating that in this region mitoses predominantly produced non-proliferative daughter cells. The results obtained were in good agreement with the model of crypt cell proliferation proposed by Cairnie, Lamerton & Steel (1965b).     

Crypt fission and crypt number in the small and large bowel of postnatal rats

The aim of this investigation was to study crypt fission, a process which may be instrumental in regulating crypt number in the intestine. Young Holtzman rats were killed at various times after parturition and samples of the small intestine and colon were removed and processed. A microdissection technique was used to separate crypts from other structures. Crypts were scored as normal or fissioning. The percentage of crypts in fission (PCF) reached peak values of 25% and 52% in the small bowel and colon, respectively, at 21 days post-parturition. From this time onward, the PCF dropped until the adult value of approximately 7% was reached in each site. During this same period, the number of crypts increased from 1.9 X 10(6) to 3.3 X 10(6) in the small bowel and 2.2 X 10(5) to 6.5 X 10(5) in the colon. Thus an inverse relationship between the percentage of crypts in fission and crypt number was found. Distribution of fissure heights in fissioning crypts did not change as the animal aged. The majority of the fissures were found in the lower 1/4 of the fissioning crypts. This suggests that as soon as the fissure extends beyond the stem cell zone, division into two crypts soon occurs.     

POPULATION DYNAMICS OF INTESTINAL EPITHELIA IN THE RAT TWO MONTHS AFTER PARTIAL RESECTION OF THE ILEUM

Sprague-Dawley rats that had been subjected 2 months previously to partial resection (10 per cent) of the small intestine and an equal number of control rats were injected with tritiated thymidine and sacrificed at intervals during the subsequent 16 hours. Segments of duodenum, jejunum and ileum were prestained by the Feulgen technique and radioautographed. The proportion of crypt cells bearing labeled nuclei, the percentage of labeled crypt cells in mitosis and the appearance of labeled crypt cells on the villi were determined. Comparison of control and resected rats showed that (a) the proportion of intestinal crypt cells incorporating thymidine was considerably greater and uniformly high throughout the shortened intestine, (b) the life cycle of crypt cells was slightly reduced, and was uniform throughout the shortened intestine, and (c) the time during which cells were retained in crypts was markedly reduced. On the basis of persistent, generalized increase in the production of crypt cells, and on prior evidence that the epithelial cells of shortened intestine continue to have a brief life span and evidence of metabolic immaturity, the existence of a humoral factor, tentatively called "intestinal epithelial growth hormone," is postulated.     

Crypt fission and crypt number in the small and large bowel of postnatal rats*

The aim of this investigation was to study crypt fission, a process which may be instrumental in regulating crypt number in the intestine. Young Holtzman rats were killed at various times after parturition and samples of the small intestine and colon were removed and processed. A microdissection technique was used to separate crypts from other structures. Crypts were scored as normal or fissioning. the percentage of crypts in fission (PCF) reached peak values of 25% and 52% in the small bowel and colon, respectively, at 21 days post-parturition. From this time onward, the PCF dropped until the adult value of approximately 7% was reached in each site. During this same period, the number of crypts increased from 1.9 × 10⁶ to 3.3 × 10⁶ in the small bowel and 2.2 × 10⁵ to 6.5 × 10⁵ in the colon. Thus an inverse relationship between the percentage of crypts in fission and crypt number was found. Distribution of fissure heights in fissioning crypts did not change as the animal aged. the majority of the fissures were found in the lower 1/4 of the fissioning crypts. This suggests that as soon as the fissure extends beyond the stem cell zone, division into two crypts soon occurs.     

HYPERSENSITIVITY REACTIONS IN THE SMALL INTESTINE

Allograft rejection of fetal intestine and graft-versus-host (GvH) disease have been used to study the effects of cell-mediated immune reactions on epithelial cell kinetics in mouse small intestine. In heterotopically transplanted isografts the cell production rate per crypt was similar to that in normally sited small intestine of the same age. However there was a six-fold increase in the rate of cell production per crypt during allograft rejection and a three-fold increase during GvH disease. Furthermore animals with GvH disease developed villous atrophy and had fewer crypts per villus than littermate controls. At the age of 19 days cell production per villus per hour was 97.5 in animals with GvH disease compared with 54.6 in controls. These results indicate that the pathological entity of ‘partial villous atrophy’evolves in two distinct phases. Phase 1, a state of increased cell turnover with crypt hyperplasia but villi of normal length precedes the development of Phase 2, true villous atrophy.     

THE PROLIFERATIVE STATUS OF INTESTINAL EPITHELIAL CLONOGENIC CELLS: SENSITIVITY TO S PHASE SPECIFIC CYTOTOXI CAGENTS

High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3-J days after a range of radiation doses indicates that a majority of the clono-genic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.     

A METHOD FOR QUANTITATION OF PROLIFERATIVE INTESTINAL MUCOSAL CELLS ON A WEIGHT BASIS: SOME VALUES FOR C57BL/6

A method for determining the number of intestinal mucosal crypts, S cells, and total proliferative cells, on a weight basis has been presented. The number of crypts was obtained (following injection of tritiated thymidine) by dividing the disintegrations per minute (dpm) per mg intestine by the dpm per crypt. Multiplication of the number of crypts per mg by the number of labeled cells per crypt (determined radioautographically) resulted in the number of S cells per mg intestine. Division of the number of S cells per mg by the fraction of proliferative cells in S (obtained by cell cycle analysis) resulted in the number of proliferative cells per mg intestine. Values for duodenum, jejunum, and ileum of male C57BL/6 mice are given.     

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.     

Colon Cryptogenesis: Asymmetric Budding

The process of crypt formation and the roles of Wnt and cell-cell adhesion signaling in cryptogenesis are not well described; but are important to the understanding of both normal and cancer colon crypt biology. A quantitative 3D-microscopy and image analysis technique is used to study the frequency, morphology and molecular topography associated with crypt formation. Measurements along the colon reveal the details of crypt formation and some key underlying biochemical signals regulating normal colon biology. Our measurements revealed an asymmetrical crypt budding process, contrary to the previously reported symmetrical fission of crypts. 3D immunofluorescence analyses reveals heterogeneity in the subcellular distribution of E-cadherin and β-catenin in distinct crypt populations. This heterogeneity was also found in asymmetrical budding crypts. Singular crypt formation (i.e. no multiple new crypts forming from one parent crypt) were observed in crypts isolated from the normal colon mucosa, suggestive of a singular constraint mechanism to prevent aberrant crypt production. The technique presented improves our understanding of cryptogenesis and suggests that excess colon crypt formation occurs when Wnt signaling is perturbed (e.g. by truncation of adenomatous polyposis coli, APC protein) in most colon cancers.     

Radiobiology and stathmokinetics of intestinal crypts associated with patches of Peyer

The stathmokinetics and radiobiology of intestinal crypts directly adjoining the lymphoid patches of Peyer, have been compared with those of non-patch-associated crypts. Patch crypts contain an additional one to two rings of cells, the Mitotic Index for the whole crypt is higher than in non-patch crypts, and the apparent cell cycle time is insignificantly lower. Using single and split doses of gamma-rays, dose-survival curves were obtained for whole intestinal crypts, from which single-cell survival curves were derived for the clonogenic cells of the crypt. For a single-hit, multitarget, model, the extrapolation numbers of the cell survival curves for patch and non-patch crypts were the same (approximately 35) but the final D0 for cells of the patch crypts was significantly higher (2.1 versus 1.7 Gy). A linear-quadratic fit gave a similar ratio of alpha/beta (approximately 10) for the two curves. For a given level of crypt depletion, the number of clonogenic cells per crypt derived by the use of equal split doses of radiation, was the same for patch and non-patch crypts. This number is a function of the dose regime employed: the higher the level of crypt depletion, the higher the derived number of cells (range 10 to 45, for non-patch crypts).     

PROLIFERATION KINETICS IN EPITHELIUM OF GUINEA-PIG COLON

The distribution of mitotic, DNA synthesizing and mature goblet cells along crypts of different length, situated in various regions of the colonic wall circumference, was determined. the distribution of proliferating cells can be approximated by a normal curve, and the distribution of mature goblet cells by an exponential curve. Some of the parameters of the distributions, different for various crypt classes, are shown to have common values when normalized by dividing them by the crypt length in a given class. the results are considered as indicating the manifestation of two levels of control of proliferation: one establishing the common pattern of the distributions along different crypts, and the second causing the differences in distributions depending on the region of the colonic wall.     

The crypt cycle. Crypt and villus production in the adult intestinal epithelium.

We propose a model for the growth of individual crypts that is able to account for the observed changes in the number of cells in crypts under normal conditions, after irradiation, and after 30% resection. Parameter values for this model are estimated both for mouse and man, and detailed predictions of crypt growth rates are made. This model does not predict a steady-state crypt size; rather it suggests that crypts grow until they bifurcate. We therefore propose a crypt cycle (analogous to the cell cycle) and present evidence that most if not all crypts in the adult mouse are cycling asynchronously and independently. This evidence consists of four experiments that indicate that branching crypts are randomly distributed over the intestinal epithelium, that the plane of bifurcation of branching crypts is randomly oriented with respect to the villus base, and that the size distribution of crypts is consistent with an expanding crypt population. We also report for the first time evidence of villus production in the adult mouse intestinal epithelium. We conclude that the crypt and villus populations in the adult mouse are not in a steady state.     

KINETICS OF TISSUE PROLIFERATION IN COLORECTAL MUCOSA DURING POST-NATAL GROWTH

The main developmental event in the colorectal mucosa during post-natal growth is a dramatic increase in the number of crypts of Lieberkühn, resulting from a longitudinal fission of pre-existing crypts. In the present study, the kinetic aspects of this process have been analysed, using extensive gland and cell counts involving the entire colon and rectum of 24 male BD IX rats distributed into four age groups. The number of crypts was found to rise from an average 4652 to 423,800 between birth and adulthood; the corresponding ratios of bifurcating glands were 13.55 and 0.67%, respectively. Crypt production attained its maximum 18 days after birth with an hourly increment of 519 units. the time spent by replicating glands in the bifurcating stage (‘fission time’) averaged 6.9–10.5 hr. The mean number of epithelial cells per crypt rose from 249 in 4-day old rats to 635 in adults. the estimated total number of epithelial cells in the colon and rectum was one million in newborns and 248 million in adults. the increment in cell number peaked 3 weeks after birth with a value of 310,000/hr. During the first few days after birth, all cells produced in the epithelium were retained. Cell loss thereafter rapidly progressed, reaching 70% of the cell production in 3-week old animals.     

HYDROXYUREA EFFECTS IN THE C3H MOUSE: I. DUODENAL CRYPT CELL KINETICS

Duodenal crypt cell kinetics in C3H mice have been studied before and after the injection of a single dose (3 mg/g body weight) of hydroxyurea (HU). This was done by autoradiographic analysis of crypt cells which had been labeled with tritiated 5-iodo-2'-deoxyuridine. This dose of HU kills the cells which are synthesizing DNA at the time of injection, inhibits DNA synthesis completely for 4–5 hr, and causes a partial synchronization of the cells when they recover from the inhibitory effects of HU. Duodenal crypt recovery is manifested by a decrease in the mean cell cycle time, an increase in the proliferating fraction, and a lengthening of the crypts. The acute cellular responses are apparently complete within 24–48 hr, but the length of the crypt has not returned to normal by 48 hr after HU administration.