Cell Tissue Kinet. (1979) 12,239-248.

CHANGES IN GROWTH KINETICS OF JEJUNAL EPITHELIUM IN MICE MAINTAINED O N AN ELEMENTAL DIET SHIRLEY LEHNERT

University Surgica I Clinic, Montreal General Hospital, Montreal Canada (Received 6 December 1977; revision accepted 26 April 1978) ABSTRACT

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. The epithelium of the small intestine has been shown to respond rapidly following alterations in the nature of the diet. Changes in intestinal proliferation have been reported to be induced by experimental modifications of the diet, ranging from starvation (Clarke. 1970; Altman. 1972) to transfer to a high bulk diet (Dowling et al.. 1967). Dietary manipulations are often employed in the management of intestinal disorders, a case in point being the use of a complete dietary formulation made of readily absorbable nutrients (elemental diet). Maintenance on an elemental diet has been reported to protect the intestinal epithelium of rats against lesions associated with the administration of 5-fluorouracil (Bounous, Hugon & Gentile, 1971b) and to increase 30-day survival in mice following 900 rad X-rays (Hugon & Bounous, 1972). Clinically an elemental diet has been used in the management of patients undergoing radiotherapy for tumours in the abdominal region (Bounous et al., 1973) and in the management of intestinal lesions induced by 5-fluorouracil and other chemotherapeutic agents (Bounous, Gentile & Hugon, 1971a; Cousineau et al.. 1973). The experiments to be described here were designed to determine whether changes in the growth kinetics of the intestinal epithelium were induced in mice maintained on the elemental diet. Correspondence: Dr Shirley Lehnert, Department of Therapeutic Radiology. Royal Victoria Hospital. 687 Pine Avenue West, Montreal, Quebec H3A l A l , Canada. 0008-8730/79/0500-0239%02.00

@ 1979 Blackwell Scientific Publications 239

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MATERIALS AND METHODS Animals Swiss ICR/HAM male mice were used in all experiments. their weight at the commencement of the experimental period being 2522.0 g. Irradiation Mice were irradiated eight at a time in a partitioned lucite box at 220 kV, 15 mA, FSD 3 7 . 5 cm. The dose rate was 54 rad/min. Diet Control animals were fed Purina mouse chow ad libitum. The test or elemental diet was Mead Johnson product 3200-AS, the composition of which is shown in Table 1, and mice were allowed unlimited access to this preparation in powdered form. Mice maintained on the elemental diet were observed to gain weight at the same rate as their chow-fed counterparts. TABLE1. Composition of the elemental diet Components Mead Johnson casein hydrolysate. Sucrose Mead Johnson medium chain triglycerides. Corn oil Olive oil Cod liver oil Vitamin mixture+ Salt mixture (Hegsted)

ghO0 g 24.9 44.7 6.9 4.3 10.4

I .3 2.5

5.0

* Mead Johnson Company of Canada Ltd. Belleville. Ontario. t Vitamin diet fortification mixture. Nutritional Biochemical Corporation. Cleveland. Ohio. Measurement of incorporation of tritiated thymidine into the jejunum Tritiated thymidine, 2.0 pCi/g (specific activity 2.0 Ci/mmol, New England Nuclear) was injected intraperitoneally. The animals were killed 30 min later by cervical dislocation and a piece of the jejunum about 1 .O cm long was removed, split longitudinally, rinsed with saline, blotted and weighed. The portions of intestine were fixed in cold Carnoy’s solution for 1-2 days, rinsed in saline and solubilized in 1.0 ml soluene (Packard Instrument Company). A toluene-based liquid scintillator was added to the vial and radioactivity was determined in a Packard liquid scintillation counter using the channels ratio method of quench correction. Measurement of cellslcrypt Two methods were used. Firstly, cells were counted on squashes prepared from dissected crypts. Portions of the jejunum about 5.0 mm long were fixed in Carnoy’s solution, hydrated through descending concentrations of alcohol, hydrolysed in 1.0 N HCI at 6OoC for 12 min and stained by the Feulgen reaction. The stained tissue was placed on a microscope slide in a

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drop of 15% acetic acid and whole crypts, separated under a dissecting microscope by probing the tissue with a fine wire, were transferred to another slide on the tip of a needle. Several crypts were thus collected in a drop of 15% acetic acid and squashed under a coverslip, the coverslip was removed by the dry ice method (Conger & Fairchild, 1953) and the slide was passed through 100% alcohol before being mounted with a coverslip. The number of cells per squashed crypt was counted using a binocular microscope with a grid mounted in the eyepiece. The second method used to determine number of cells/crypt was counting crypt cells on histological sections. In transverse sections of jejunum, crypts were selected where the plane of the section passed longitudinally through the centre of the crypt and the number of cells in the column along the length of the crypt was counted. The number of columns was measured by counting the number of cells around the periphery of the crypt in sections which were cut tangentially to the lumen to the intestine. The number of cells/crypt column multiplied by the number of columns gave the number of cells/crypt.

Measurement of villus length This was done by counting the number of cells along the length of the villus in sections where the plane of the section clearly passed through the tip and the base of the villus. Autoradiography Autoradiographs were prepared of squashes of crypt cells prepared as described above. For determination of the labelling index, mice were injected with 50 pCi tritiated thymidine 30 min prior to death. Autoradiographs of squashes were made using Kodak NTB-2 liquid emulsion. They were exposed for 10 days at 4OC, developed, mounted with coverslips and the percentage of labelled cells scored. For determination of duration of phases of the cell cycle by the percentage of labelled mitoses method, mice were injected intraperitoneally with 50 pCi of tritiated thymidine. At hourly intervals thereafter, pairs of mice were killed and autoradiographs of crypt squashes were prepared. The percentage of labelled mitoses were determined by inspection of 200 mitotic cells on each slide. Measurement of intestinal cell transit time The procedure of Sigdestad, Hagemann & Lesher (1972) was used. Mice were injected with 2.0 pCi/gm tritiated thymidine every 6 hr for 70 hr. 30 min after each injection, pairs of mice were killed and the radioactivity of duplicate samples of the jejunum was assayed as described above. If d/min per mg weight of jejunum is plotted against time, the curve shows an ascending portion (as crypt cells incorporate tritiated thymidine) followed by a plateau (when incorporation of tritiated thymidine into cells in the crypt is balanced by loss of radioactivity as cells are extruded at the tips of the villi). The intersection of the regression lines fitted to these two components of the curve represents the cell transit time. RESULTS

Changes in numbers of villus cells and in proliferative rate of the jejunum in dietfed mice The most apparent change induced in the intestine of diet-fed mice is an increase in the number of cells along the length of the villus. In Fig. 1 the number of cells per villus column is shown at various times after commencement of feeding the elemental diet. A maximum

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number of cells/column is seen in mice maintained on the diet for 2 days, for longer feeding times the number declines slightly until after 6 days of feeding it is stable at a value 15-20% above that of chow-fed controls. Also shown in Fig. 1 are the changes in proliferative activity in the jejunum which occur following transfer to mice from chow to the elemental diet. The rate of incorporation of tritiated thymidine into the jejunum is reduced in mice maintained on the elemental diet for 1 day and is minimal at 2 days. Values close to those seen in chow-fed controls are seen in mice fed the diet for 4, 5 and 6 days. However, by 8 days after commencement of feeding, the level of tritiated thymidine incorporation into the jejunum is 80% that seen in chow-fed controls and this level is maintained for as long as the diet

3

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9

1

0

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1

2

Time on dlet (days)

FIG. I . (0) Number of villus cells/column. and (D) incorporation of tritiated thymidine into the jejunum, at various times after transferring to an elemental diet. Values expressed as percentage of those in chow-fed controls. The height of each column represents mean (2s.e.m.) of values from five to six mice. Control (10Oo~o) values were arrived at as follows: villus celldcolumn from the mean of counts of villus cells/column in six mice. with five villi counted/mouse. Incorporation of tritiated thymidine from the mean of incorporation d/min per mg wet weight) for eight chow-fed mice killed at the beginning of the experimental period.

continues to be fed. Thus in mice fed the elemental diet for prolonged periods an 'equilibrium' is reached when the number of cells in the villus is approximately 20% greater and the rate of proliferation is approximately 2Oohless than that seen in chow-fed controls.

Mechanism by which the prolijerative capacity of the crypt is reduced This was investigated in mice maintained on the elemental diet for 9 or 10 days, at which time the number of villus cells, and the rate of proliferation, had stabilized at levels characteristic of diet-fed mice. The total number of cells in the crypt was determined by counting cells on either squashes or sections. Results of both procedures show the number of cells per crypt in diet-fed mice to be reduced by approximately 30% compared with chow-fed mice (Table 2). The labelling index of crypt cells was determined on autoradiographs of crypt squashes and no significant difference was seen between diet-fed mice and chow-fed controls. Pcrcentage labelled mitosis curves for diet-fed and chow-fed mice are shown in Fig. 2. From these curves the generation time (T,) and the DNA synthetic time (T, ) were derived

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TABLE2. Effects of elemental diet on crypt cell production ~

~~

Time on diet (days)

-~ Cells/crypt (from squashes) Celldcrypt (from sections) ~

Labelling index? T, (hr) T, (hr) Proliferative fraction Proliferative ceWcrypt

~

542 ? 44 535 (22.3 _+ 0.48 x 24.0 & 0.7)* 0.41 5 0.412 12-75 7.5 0.69 375

398 _+ 24 366 (18.6 _+ 0.39 x 19.7 k 0.59) 0.364 5 0.013 12.75 7.5 0.62 245

* Number of cells/column x number of columns. Three mice were used to count cells/crypt, and five crypts were counted from each mouse. t Six mice were used for determination of labelling index, and 5000 cells were counted on each slide.

for cells of the jejunal crypt. Tg was obtained by measuring the time elapsed between the 50% point on the first and second ascending limbs of the curve and was 12.75 hr for both chow-fed and diet-fed mice. T, is the time between the 50% points on the ascending and

Time offer [3Hlthymidine inlection (hr)

FIG. 2. (a) Percentage of labelled mitoses in control mice and (b) mice maintained on elemental diet for 9 days prior to experiment. Two mice were killed at each time interval and 200 mitotic cells were scored on each slide.

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descending limbs of the first portion of the curve and was 7.5 hr for both groups of mice. The proliferative fraction for the whole crypt was calculated from the equation (Al-dewachi et al., 1974): proliferative fraction

= LIexp/LItheor.

(1)

Where LI,,, is the observed labelling index and LItheoris the theoretical labelling index derived from the ratio TJT,. The product of the proliferative fraction and the number of cells per crypt gives the number of proliferative cells/crypt. In mice maintained on the elemental diet for 9 days the number of proliferative cells/crypt was about 65% of the number in chow-fed mice (Table 2). Villus cell transit time in chowfed and dietfed mice Since the number of villus cells/column is increased in mice maintained on the elemental diet the next experiment was designed to determine whether there is a concomitant increase in the transit time of the epithelial cell from the base to the tip of the villus. The technique used was that described by Sigestad et al. ( 1 972) and the results of one experiment are shown in Fig. 3. For chow-fed mice, the cell transit time, indicated by the point at which lines fitted to the two components of the curve intersect, is 44 hr (Fig. 3a). For diet-fed mice (the experiment was carried out from the eighth to the eleventh day of feeding the diet), the cell

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36

48

60

I 72

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84

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Time ( h r l

FIG. 3. Incorporation of tritiated thymidine into jejunum of mice injected at intervals of 6 h. Points represent mean of values of duplicate samples from two mice killed at each time interval. (a) Control mice: (b) mice maintained on elemental diet from 9 days prior to commencement of, and during, the experiment.

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transit along the villus is 58 hr (Fig. 3b). Further experiments are required to determine whether a difference of this magnitude in villus cell transit time is consistently observed in diet-fed mice.

Response to irradiation of control and dietfed mice To determine whether changes induced in intestinal epithelial cell kinetics of diet-fed mice influenced the capacity of the epithelium of these mice to respond to depletion of the crypt compartment, the rate of proliferation of the jejunum following whole body X-irradiation was studied (Fig. 4). Following a dose of 1100 rad to the whole body, incorporation of tritiated thymidine into the jejunum fell to a minimum within 1 day of irradiation, remained low the following day then increased, reaching a maximum at 4 days irradiation, at which time the

T

7,

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Time after irradiation (days1

FIG.4. Incorporation of tritiated thymidine into jejunum of mice at various times after 1100 rad whole body irradiation. Six mice were killed in each group, with vertical bars representing the Control mice; (0)diet-fed mice. standard errors. (0)

rate of incorporation exceeded that seen prior to irradiation. The same pattern was observed in both chow-fed and diet-fed mice, but in diet-fed animals the initiation of the increase in tritiated thymidine incorporation following irradiation was delayed by approximately 16 h and the overshoot in tritiated thymidine incorporation was not seen.

DISCUSSION The earliest response of the jejunum of the mouse to ingestion of the elemental diet is an increase in the number of cells per column in the villus. The maximum number of cells per villus column (133% of control) is seen within 2 days of commencement of feeding the diet, the number subsequently declines to a value, approximately 120% of control, which is maintained for as long as the mice continue to ingest the diet. The reason for the increased cellularity of the villus is not known: it is possible, however, that it results from reduced attrition of cells close to the tips of the villi. Factors which might influence the rate at which cells are exfoliated from the villus tips include the bulk and mechanical characteristics of the gut contents, the nature of intestinal secretions into the gut lumen, and the nature of the

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intestinal flora. It has been shown that changes produced in the intestine of dogs fed the elemental diet include a quantitative reduction of flora, and of bulk (Bounous et al., 1967), while a decrease in the dipeptidase activity recoverable from the intestinal mucosa has been demonstrated in rats fed the elemental diet (Bounous et al., 1971b). Thus in the diet-fed mouse it is possible that several changes may occur which are conducive to prolongation of the life span of the villus cell. Concomitant with the changes in the cellularity of the villus are changes in proliferative activity of the crypt. The incorporation of tritiated thymidine in the jejunum is at a minimum within 2 days after commencing the diet, and after some fluctuation it reaches a level of about 20% below that of controls and remains constant at this level for as long as the mouse is maintained on the diet. The reduction in crypt cell production in diet-fed mice was found to result from a reduction in the total number of cells in the crypt with no change in either proliferative fraction or the duration of the phases of the cell cycle. Changes in crypt cell production have also been observed to occur in response to a variety of other natural conditions or experimental manipulations. and the mechanisms by which these changes are produced are similarly varied. In circumstances where crypt cell production is increased, the change may be brought about by an increase in the total number of crypt cells with no increase in the proliferative fraction, and this process occurs in a segment of ileum transposed to the jejunum (Rijke, Hanson & Plaisier, 1977). Crypt enlargement is also seen in lactating mice (Harding & Cairnie, 1975) and following partial resection of the intestine (Hanson & Osborne, 1971). In these two cases, however, a concomitant shortening of the cell cycle time in the crypt was observed. An increase in crypt cell production was also found after experimental reduction of the villus cell population (Rijke et al.. 1976). In this case, however, the increase in crypt cell production was produced by an increase in the relative size of the proliferative cell compartment in the crypt. whereas crypt size did not increase. A reduction in crypt cell production is seen in germ-free animals and here an analogy may be drawn with diet-fed mice, since reduced crypt cell production is seen in conjunction with an increased number of villus cells/column. Abrams, Bauer & Sprinz (1963) observed a reduction in the size of the crypt in germ-free mice while Lesher. Walburg & Sacher (1964) found an increase in the generation time of crypt cells from germ-free mice resulting from an increase in duration of both G, and S. Galjaard, van der Mier Fieggen & Giesen (1972), working with rats, found the percentage of labelled crypt cells to be the same in conventional and germ-free animals. Thus it seems likely the reduction of crypt cell production in germ-free animals is achieved by reduction in the total number of cells in the crypt and possibly by changes in growth kinetics among those cells. It has been suggested that in some cell renewal systems the size of the functional compartment may exert a type of feedback control on the proliferative activity of the stem cell compartment. Such feedback control has been described for the skin (Bullough & Laurence, 1964; Bullough, 1967) and haematopoietic tissue (Rytomaa & Kiviniemi, 1968; Kivilaakso & Rytomaa. 1971), and there is also strong evidence that in the intestine the size of the pool of functional villus cells is involved in regulation of proliferative activity in the crypt (Galjaard et al., 1972: Rijke et al., 1976). Rijke et al. (1977) have speculated that crypt cell production is controlled by two different mechanisms, one of which regulates the size of the crypt, while the other determines the relative size of the proliferative fraction, and that the latter mechanism is characteristic of the feedback control of crypt proliferation exerted by functional villus cells. There is a close temporal relationship between the increase in the cells/villus column and

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the decrease in crypt cell production seen when chow-fed mice are transferred to the elemental diet. However, the fact that these changes appear to occur simultaneously rather than in sequence does not lend credence to the speculation that the number of villus cells is involved in regulating the rate of cell production in the crypt. Moreover, the means by which crypt cell production is reduced in diet-fed mice, i.e. by a reduction in the total number of crypt cells, with no apparent change in the kinetics of these cells, is, at least according to the hypothesis of Rijke et al. (1977), not characteristic of the means by which feedback regulation would work. It seems likely that changed nature of the gut contents following transfer of the mouse to the elemental diet constitutes the stimulus for the change in the rate of crypt cell production which is coincidental with, but not regulated by, the change in the cellularity of the villus. The response of diet-fed mice to irradiation may, however, be described in terms of regulatory negative feedback mechanism, and here a clear parallel between germ-free and diet-fed animals is seen. Galjaard et al. (1 972) reported that in germ-free rats the mean height of the villus was 105 cells/column compared with seventy-six cells/column in conventional rats of the same strain. An inverse relationship was observed between the number of cells/villus column and the percentage of labelled cells in the crypt in irradiated germ-free and conventional rats. Following single doses of radiation, temporary expansion of the pool of proliferating cells started in conventional rats 36-48 hr after irradiation and 60 hr after irradiation in germ-free animals. In the diet-fed animals described here the mean number of cells per villus column increased from sixty to seventy-two and, following irradiation, the initiation of compensatory proliferation in the crypt occurs approximately 16 hr later than in chow-fed mice. The results from both germ-free and diet-fed mice support the hypothesis that one factor regulating the timing of the proliferative response to irradiation is the time required post-irradiation to deplete the villus cell population to a level which will trigger increased proliferative in the crypt. These findings demonstrate the rapid adaptation of the intestinal epithelium to changes in the nature of the diet. Further study of the influence of dietary factors on the renewal rate of the intestinal epithelium will be of interest both from the stand-point of elucidating the controlling factors and because dietary manipulations are so frequently employed in the management of intestinal disorders. ACKNOWLEDGMENTS

These investigations were supported by the John A. Hartford Foundation. I am indebted to Claudette Labelle and Kathy Key for technical assistance. REFERENCES ABRAMS,G.D., BAUER,H. & SPRINZ, H. (1963) Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. A comparison of gern-free and conventional mice. Lab. Invest. 12 355. H., WRIGHT,N.A., APPLETON,D. & WATSON,A.J. (1974) The cell cycle time in the rat jejunal AL-DEWACHI, mucosa. Cell Tissue Kinet. I, 587. ALTMAN,G.G. (1972) Influence of starvation and refeeding on the mucosal size and epithelial renewal in the rat small intestine. Amer. J. Anat. 133, 391. BOUNOUS,G., GENTILE, J.M. & HUGON,J. (1971a) Elemental diet in the management of intestinal lesions produced by 5-fluorouracil in man. Canad. J. Surg. 14,3 12. G., HUGON,J. & GENTILE,J.M. (1971b) Elemental diet in the management of lesions produced by 5BOUNOUS, fluorouracil in rats. Canad. J. Surg. 14,298.

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BOLJXOLJS. G.. SLJTHERLAND. N.G., MCARDLE,A.H. & C U R D F.N. , (1967) Prophylactic use of 'elemental' diet in experimental hemorrhagic shock and intestinal ischemia. A n n . Surg. 166. 3 12. BOUNOUS. G.. TAHAN.W.. SHLJSTER. J.. GOLD.P.. COLJSINEALJ. L.. ROCHAN.M. & LEBEL. E. (1973) The use of an elemental diet during abdominal radiation. Clin. Res. 21. 1066. BULLOLJGH. W.S. (1967) The Ecolution of Differentiation.Academic Press. London. BLJLLOLJGH. W.S. & LAURENCE. E.B. ( I 964) The production of epidermal cells. Symp. 2001.SOC.Lond. 12, 1. CLARKE.R. (1970) The effect of starvation upon the mucosal architecture and cell production rate in the small intestine of the rat. J . Anal. 107. 384. L.M. (1953) A quick-freeze method for making smear slides permanent. Slain CONGER,A.D. & FAIRCHILD, Technol. 28. 28 1. COCISINEAU. L.. BOLJNOLJS. G.. ROCHON.M.. SHUSTER. J.. GOLD.P. & TAHAN.W. (1973) The use of an elemental diet during treatment with anticancer agents. Clin. Res. 21. 1066. E.O.. LAWS.J.W. & BOOTH.C.C. (1967) The intestinal response to high bulk feeding DOWLING. R.H.. RIECKEN. in the rat. Clin. Sci. 32. 1. GAUAARD. H., MEER-FIEGGEN, W. V A N DER & GIESEN,J. (1972) Feedback control by functional villus cells on cell proliferation and maturation of intestinal epithelium. Exp. Cell Res. 73. 197. HANSON.W.R. & OSBORNE.J.W. (1971) Epithelial cell kinetics in the small intestine of the rat 6 0 days after resection of 70 percent of the ileum and jejunum. Gastroenterology, 60. 1087. A.B. (1975) Changes in intestinal cell kinetics in the small intestine of lactating mice. HARDING. J.D. & CAIRNIE, Cell Tissue Kinet. 8. 135. G. (1972) Elemental diet in the management of the intestinal lesions produced by HLJGON.J.S. & BOLJNOLJS. radiation in the mouse. Canad. J . Surg. 15. 18. KIVILAAKSO. E. & RYTOMAA. T.(1971) Erythrocyte chalone. a tissue specific inhibitor of cell proliferation in the erythron. Cell Tissue Kinet. 4. 1. H.E. & SACHER. G.A. (1964) Generation time in the duodenal crypt cells of germ-free and LESHER.S.. WALBURG. conventional mice. N a m e (Lond.). 202. 884. RIJKE.R.P.C.. HANSON.W.R. & PLANER.H.M. (1977) The effect of transposition to jejunum on epithelial cell kinetics in an ileal segment. Cell Tissue Kiner. 10. 399. J.W. ( I 976) Effect of ischemic villus cell damage on RUKE.R.P.C.. HANSON.W.R., PLAISIER. H.M. & OSBORNE. crypt cell proliferation in the small intestine. Evidence of a feedback control mechanism. Gastroenterology, 71. 786. K. (1968) Control of granulocyte production. 11. Mode action of chalone and antiRYTOMAA,T. & KIVINIEMI. chalone. Cell Tissue Kinet. 1, 341. SIGDESTAD. C.P.. HAGEMANN, R.F. & LESHER.s. (1972) A new method for measuring intestinal cell transit time. Gastroenterology. 58. 47.

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

Cell Tissue Kinet. (1979) 12,239-248. CHANGES IN GROWTH KINETICS OF JEJUNAL EPITHELIUM IN MICE MAINTAINED O N AN ELEMENTAL DIET SHIRLEY LEHNERT Univ...
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